Kava derived therapeutic compounds and methods of use thereof

ABSTRACT

Certain embodiments of the invention provide a composition comprising at least two compounds selected from the group consisting of dihydromethysticin, methysticin, dihydrokavain, kavain, desmethoxyyangonin and 11-methoxyyangonin, wherein the composition is substantially free of flavokawain B. Certain embodiments of the invention also provide a method for treating or preventing cancer in a mammal (e.g., a human) in need of such treatment comprising, administering to the mammal a carrier and a compound selected from the group consisting of dihydromethysticin, methysticin, dihydrokavain, kavain, desmethoxyyangonin and 11-methoxyyangonin, wherein the compound is substantially free of other kava extract components.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/902,635 filed Nov. 11, 2013 and U.S. Provisional PatentApplication No. 61/904,791 filed Nov. 15, 2013, the entirety of whichare incorporated herein by reference.

GOVERNMENT FUNDING

This invention was made with government support under R01-CA142649awarded by the National Cancer Institute/National Institutes of Health.The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Lung cancer is the leading cause of malignancy-related mortality becauseof its high incidence and the lack of effective treatments. Sincetobacco usage contributes to 85-90% of its development, tobaccocessation is the most straightforward strategy for reducing lung cancerincidence and mortality. However, because of the addictive nature ofnicotine in tobacco, limited progress has been achieved in reducingtobacco usage. An alternative approach is to block or slow down tobaccocarcinogen-induced lung cancer development via chemoprevention (Hecht etal., Nat. Rev. Cancer 2009; 9:476-88). Although a number of compoundshave been identified as potential chemopreventive agents against lungtumorigenesis in animal models, their moderate in vivo efficacy leavesample room for improvement and introduces significant challenges forclinical application/evaluation. In addition, there are very limitedsuccesses in cancer chemoprevention relative to cancer therapy.Therefore, there is currently an unmet need for additional agents thatare useful for treating or preventing cancer.

SUMMARY OF THE INVENTION

Certain embodiments of the invention provide a method, comprising:

-   -   a) combining an ethanolic kava extract and silica gel to provide        a mixture;    -   b) evaporating the mixture to provide a silica gel having kava        residue adsorbed thereon;    -   c) loading the silica gel having kava residue adsorbed thereon        on a chromatography column to provide a kava-adsorbed silica gel        column;    -   d) eluting the kava-adsorbed silica gel column with a solvent        system to provide a first kava extract fraction, a second kava        extract fraction, and a third kava extract fraction, wherein the        first kava extract fraction consists essentially of non-polar        compounds, including flavokawains, the second kava extract        fraction consists essentially of kavalactones and flavanones,        and the third kava extract fraction consists essentially of        polar compounds.

Certain embodiments of the invention provide a second kava extractfraction prepared by

a method described herein.

Certain embodiments of the invention provide a composition comprising11-methoxyyangonin and/or flavanone:

and at least one compound selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain anddesmethoxyyangonin, wherein the composition is substantially free offlavokawain B and/or flavokawain A.

Certain embodiments of the invention provide a composition comprising atleast one compound selected from the group consisting ofdihydromethysticin, wherein the weight percent of dihydromethysticin inthe composition is about 20 to 99%; methysticin, wherein the weightpercent of methysticin in the composition is about 10 to 99%;dihydrokavain, wherein the weight percent of dihydrokavain in thecomposition is about 40 to 99%; kavain, wherein the weight percent ofkavain in the composition is about 40 to 99%; desmethoxyyangonin,wherein the weight percent of desmethoxyyangonin in the composition isabout 30 to 99%; and 11-methoxyyangonin, wherein the weight percent of11-methoxyyangonin in the composition is about 20 to 99%.

Certain embodiments of the invention provide a kava extract comprising11-methoxyyangonin and/or flavanone:

and at least one compound selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain anddesmethoxyyangonin, wherein the extract is substantially free offlavokawain B and/or flavokawain A.

Certain embodiments of the invention provide a kava extract consistingessentially of dihydromethysticin, 11-methoxyyangonin,desmethoxyyangonin, dihydrokavain, kavain, methysticin and flavanone:

Certain embodiments of the invention provide a pharmaceuticalcomposition comprising a composition or kava extract as described hereinand a pharmaceutically acceptable carrier.

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal in need of such treatment comprising,administering to the mammal dihydromethysticin and a carrier, whereinthe dihydromethysticin is substantially free of other kava extractcomponents.

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal in need of such treatment comprising,administering to the mammal methysticin and a carrier, wherein themethysticin is substantially free of other kava extract components.

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal in need of such treatment comprising,administering to the mammal dihydrokavain and a carrier, wherein thedihydrokavain is substantially free of other kava extract components.

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal in need of such treatment comprising,administering to the mammal kavain and a carrier, wherein the kavain issubstantially free of other kava extract components.

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal in need of such treatment comprising,administering to the mammal desmethoxyyangonin and a carrier, whereinthe desmethoxyyangonin is substantially free of other kava extractcomponents.

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal in need of such treatment comprising,administering to the mammal 11-methoxyyangonin and a carrier, whereinthe 11-methoxyyangonin is substantially free of other kava extractcomponents.

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal in need of such treatment comprising,administering to the mammal a composition or kava extract as describedherein.

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal dihydromethysticin anda carrier, wherein the dihydromethysticin is substantially free of otherkava extract components.

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal methysticin and acarrier, wherein the methysticin is substantially free of other kavaextract components.

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal dihydrokavain and acarrier, wherein the dihydrokavain is substantially free of other kavaextract components.

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal kavain and a carrier,wherein the kavain is substantially free of other kava extractcomponents.

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal desmethoxyyangonin anda carrier, wherein the desmethoxyyangonin is substantially free of otherkava extract components.

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal 11-methoxyyangonin anda carrier, wherein the 11-methoxyyangonin is substantially free of otherkava extract components.

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal a composition or kavaextract as described herein.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIG. 1. Characterization of the effect of kava and kava fractions on DNAadducts induced by NNK in the lung of A/J mice (n=3 each group):*p<0.05; **p<0.01; ***p<0.001. A. The amount of DNA adducts at differenttime points after NNK treatment; NNK alone:

; NNK+kava:

). B. Relative amount of DNA adducts in NNK+kava treatment group atdifferent time points after NNK treatment (the amount with kavatreatment normalized to that induced by NNK alone at the same timepoint). C. The amount of DNA adducts with different kava fractiontreatment 24 h after NNK treatment. D. Relative amount of DNA adducts bydifferent kava fractions normalized to that induced by NNK alone at the24 h time point.

FIG. 2. Two metabolic activation pathways of NNK leading to differentmethylated—vs. 4-(3-pyridyl)-4-oxobut-1-yl (pob)-DNA adducts. Solidblocks indicate measured reduction in different classes of DNA adducts,as exemplified by O⁶-mG vs. 7-pobG, O²-pobdT, and O⁶-pobdG. Dashedblocks indicate hypothetical points of action by kava chemicals or theirmetabolites: (i) to differentially inhibit cytochrome p450isoform-mediated NNK metabolic activation or (ii) directly react withNNK methylene hydroxylation metabolites as chemical traps.

FIG. 3. Compounds in Fraction B, including dihydromethysticin andmethysticin.

FIG. 4. Study design for evaluating the chemopreventive efficacy ofdifferent kava treatment regimens against NNK-induced lung adenomaformation in A/J mice. Beginning at the age of 6-7 weeks, groups offemale A/J mice (Groups 2-9) were treated by two dosages of NNK (100 and67 mg/kg bodyweight on Day 7 and Day 14 respectively) via i.p. injectionin 0.1 mL saline. Mice in Group 1 were given two doses of saline (0.1 mLeach) on Day 7 and Day 14. Mice in Groups 1 and 2 were maintained onstandard diet treatment for the duration of the experiments. Mice ingroups 3-9 were given a diet supplemented with kava at a dose of 5 mg/gdiet according to the following schedules: Group 3 (Day 1-Day 14); Group4 (Day 1-Day 21); Group 5 (Day 1-Day 119); Group 6 (Day 15-Day 119);Group 7 (Day 15-Day 28); Group 8 (Day 22-Day 119) and Group 9 (Day29-Day 119). The study was terminated on Day 119.

FIG. 5. Representative photomicrographs H&E-stained sections of lungs(n=4 in each group) from negative control mice (A), mice with NNK alone(B), and mice with NNK plus kava at a dose of 5 mg/g of diet (C).

FIG. 6. ¹H-NMR spectra of different kava fractions and the mass balanceof each fraction. I. Kava; II. Fraction A; III. Reconstituted kava fromFractions A, B, and C; IV. Fraction B; V, Comparison between Kava(bottom line) and reconstituted kava (top line) from Fractions A, B, andC; VI. Fraction C; VII. Mass balance of each fraction.

FIG. 7. HPLC traces of traditional kava, kava from Gaia Herbs,reconstituted kava, and Fractions A, B, and C.

FIG. 8. Chemicals isolated from kava and their natural abundance.

FIG. 9. Genito-urinary track weight (16 weeks of age) (excluding 1 tumorbearing mouse). For each group, the bar on the left represents wildtypemice and the bar on the right represents TRAMP mice.

FIG. 10. Prostate Weight (16 weeks of age). For each group, the bar onthe left represents wildtype mice and the bar on the right representsTRAMP mice.

FIG. 11. GU Weight (28 weeks of age) (excluding tumor-bearing mice). Foreach group, the bar on the left represents wildtype mice and the bar onthe right represents TRAMP mice.

FIG. 12. Prostate Weight (28 weeks of age) (excluding tumor-bearingmice). For each group, the bar on the left represents wildtype mice andthe bar on the right represents TRAMP mice.

FIG. 13. The effect of dihydromethysticin and methysticin on reducingtumor volume of lung tumors inoculated by human lung H2009 cancer celllines.

FIG. 14. The effect of Fractions A, B, and C on reducing tumor volume oflung tumors inoculated by human lung A549 cancer cell lines. In thebottom panel (B), the bars represent the following, from left to right:Vehicle Control, Kava Fraction A, Kava Fraction B and Kava Fraction C.

FIG. 15. The effect of kava and Fraction B on reducing tumor volume oflung tumors inoculated by human lung A549 cancer cell lines and safety.

FIG. 16. Metabolic pathways of NNK leading to formation of DNA adductsand hypothetic points of action by DHM for its selective reduction in asubset of NNK-induced DNA damage in A/J mouse lung—preferentialinhibition of NNAL activation via hydroxylation (A) or enhancingdetoxification of NNAL via glucuronidation (B).

FIG. 17. Characterization of natural kavalactones on NNK-induced DNAdamage in A/J mouse lung tissues. A, Chemical structures of five naturalkavalactones isolated from Fraction B. B, Their effects on NNK-inducedPOB adducts and O⁶-mG adduct. Comparison was made with the NNK treatmentgroup by Dunnett's test when ONE-WAY ANOVA was statisticallysignificant. n=3 each group. *p<0.05 and **p<0.01.

FIG. 18. Characterization of the effect of different agents onNNK-induced DNA damage in A/J mouse lung tissues. A, Dose-responseeffect of natural (+)-DHM on NNK-induced O⁶-mG (a) and three POB adducts(b-d) in comparison to kava, (+)-DHK and synthetic (±)-DHM. B,Dose-response effect of natural (+)-DHM on NNK-induced 7-mG (a) andthree PHB adducts (b-d) in comparison to kava, (+)-DHK and synthetic(±)-DHM. C, Percentage of different DNA adducts from mice treated with(+)-DHM (1 mg/g of diet) relative to that from NNK-treated control mice.For A and B, comparison was made with NNK treatment group by Dunnett'stest when ONE-WAY ANOVA was statistically significant. n=3 each group.*p<0.05 and **p<0.01. For C, ONE-WAY ANOVA was not statisticallysignificant.

FIG. 19. Bodyweight and major organs from the control mice and mice with(+)-DHM exposure at a dose of 0.5 mg/g of diet at Week 17. p values weregiven with comparison between the control group (n=5) and (+)-DHMtreatment group (n=10) using a two-tailed Student t-test.

FIG. 20. Body weight and food consumption data for A/J mice from controland (+)-DHM (0.5 mg/g of diet) treated group. A, Weekly time-course ofbodyweight changes. B, Average daily-food consumption (disappearance)estimated weekly. The weekly bodyweights of the control and (+)-DHMtreated mice were analyzed by a two-tailed Student t-test, and none ofthe comparisons were statistically significant.

FIG. 21. Clinical chemistry results of serum samples from the controlmice and mice with (+)-DHM exposure at a dose of 0.5 mg/g of diet. A, 8weeks. B, 17 weeks. p values were given when possible with comparisonbetween the control group (n=5) and the (+)-DHM treatment group (n=10)using a two-tailed Student t-test.

FIG. 22. Hematological results of blood samples from control mice andmice with exposure to (+)-DHM at a dose of 0.5 mg/g of diet for 17weeks. p values were given when possible with comparison between thecontrol group (n=4) and the treatment group (n=10) using two-tailedStudent t-test.

FIG. 23. The effect of 14-week daily kava treatment (500 mg/kgbodyweight) via gavage on mouse serum ALT (A) and AST (B). p values weregiven with comparison between the control group (n=4) and kava treatmentgroup (n=4) using the two-tailed Student t-test.

FIG. 24. The effect of 3-day daily kava treatment (500 mg/kg bodyweight)via gavage on mouse serum ALT and AST and liver lesions with/withoutAPAP treatment (800 mg/kg bodyweight) via gavage. A. Serum ALT and AST.B. The number of mice with different grades of liver lesions. C. Therelationships among serum ALT, AST and the grades of liver lesions. ForA, comparisons were made with APAP treatment group by Dunnett's testwhen ONE-WAY ANOVA was statistically significant (n=8−15).

FIG. 25. Chemical structures of flavokawains A, B, anddihydromethysticin.

FIG. 26. The effect of 3-day daily DHM (37.5 mg/kg) or FKB (11.5 mg/kg)via gavage on mouse serum ALT and AST with/without APAP treatment.Comparisons were made with APAP treatment group by Dunnett's test whenONE-WAY ANOVA was statistically significant (n=6−15).

FIG. 27. The dose-response effect of 3-day daily FKA and FKB via gavageon mouse serum ALT, AST and livers with/without APAP treatment. A & B.Serum ALT and AST. Comparisons were made with APAP treatment group byDunnett's test when ONE-WAY ANOVA was statistically significant (n=5).C. Serum level of ALT and AST from the dead mouse in the high-dose FKAand FKB group with APAP co-treatment. D. Photomicrographs of H&E-stainedlivers from a control mouse (Panel A) and a mouse treated with FKA andFKB plus APAP (Panel B). Note extensive karyorrhexis (arrow) reflectingacute necrosis of hepatocytes (increased eosinophilia) in mouse treatedwith FKA, FKB and APAP (Panel B).

FIG. 28. The dose-response effect of dihydromethysticin on reducing2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (A) andbenzo(a)pyrene-induced DNA damage (B) in cell culture Hepe1c1c7 cells.

Table 1. Effect of different kava treatment schedules on lung tumorincidence and multiplicity induced by NNK in A/J mice at 119 day endpoints.

Table 2. Effect of kava on lung tumor incidence and multiplicity inducedby NNK in A/J mice at 25 weeks (175 days) and 34 week (238 days) timepoint.

Table 3. Effect of bolus kava via daily gavage on lung tumor incidenceand multiplicity induced by NNK in A/J mice.

Table 4. Dose-response effect of kava and different kava fractions onlung tumor incidence and multiplicity induced by NNK in A/J mice.

Table 5. Experimental design for prostate cancer prevention with TRAMPmodels.

Table 6. Prostate tumors at the age of 28 weeks.

Table 7. Prostate tumor weight (g) and lobe location (VP=ventralprostate lobe).

Table 8. Effect of different agents on lung tumor incidence andmultiplicity induced by NNK in A/J mice.

DETAILED DESCRIPTION

Certain embodiments of the invention provide a composition comprising atleast two compounds selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin, wherein the composition issubstantially free of flavokawain B and/or flavokawain A.

In certain embodiments, the at least two compounds aredihydromethysticin and methysticin.

In certain embodiments, the at least two compounds aredihydromethysticin and dihydrokavain.

In certain embodiments, the at least two compounds aredihydromethysticin and kavain.

In certain embodiments, the at least two compounds are methysticin anddihydrokavain.

In certain embodiments, the at least two compounds are methysticin andkavain.

In certain embodiments, the at least two compounds are dihydrokavain andkavain.

In certain embodiments, the composition comprises at least threecompounds selected from the group consisting of dihydromethysticin,methysticin, dihydrokavain, kavain, desmethoxyyangonin and11-methoxyyangonin, wherein the composition is substantially free offlavokawain B and/or flavokawain A.

In certain embodiments, the at least three compounds aredihydromethysticin, methysticin and dihydrokavain.

In certain embodiments, the at least three compounds aredihydromethysticin, methysticin and kavain.

In certain embodiments, the at least three compounds aredihydromethysticin, dihydrokavain and kavain.

In certain embodiments, the at least three compounds are methysticin,dihydrokavain and kavain.

In certain embodiments, the composition comprises at least fourcompounds selected from the group consisting of dihydromethysticin,methysticin, dihydrokavain, kavain, desmethoxyyangonin and11-methoxyyangonin, wherein the composition is substantially free offlavokawain B and/or flavokawain A.

In certain embodiments, the composition comprises at least fivecompounds selected from the group consisting of dihydromethysticin,methysticin, dihydrokavain, kavain, desmethoxyyangonin and11-methoxyyangonin, wherein the composition is substantially free offlavokawain B and/or flavokawain A.

In certain embodiments, the composition comprises dihydromethysticin,methysticin, dihydrokavain, kavain, desmethoxyyangonin and11-methoxyyangonin, wherein the composition is substantially free offlavokawain B and/or flavokawain A.

In certain embodiments, the composition further comprises flavanone:

In certain embodiments, the composition is substantially free offlavokawain B.

In certain embodiments, the composition is substantially free offlavokawain A.

In certain embodiments, the composition is substantially free of bornylester of 3,4-methylenedioxy cinnamic acid:

In certain embodiments, the composition is substantially free of bornylester of cinnamic acid:

In certain embodiments, the composition is substantially free offlavanone:

In certain embodiments, the composition is substantially free ofpinostrobin.

Certain embodiments of the invention provide a composition comprising11-methoxyyangonin and/or flavanone:

and at least one compound selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain anddesmethoxyyangonin, wherein the composition is substantially free offlavokawain B and/or flavokawain A.

In certain embodiments, the at least one compound is dihydromethysticin.

In certain embodiments, the composition comprises at least two compoundsselected from the group consisting of dihydromethysticin, methysticin,dihydrokavain, kavain and desmethoxyyangonin, wherein the composition issubstantially free of flavokawain B and/or flavokawain A.

In certain embodiments, the composition comprises at least threecompounds selected from the group consisting of dihydromethysticin,methysticin, dihydrokavain, kavain and desmethoxyyangonin, wherein thecomposition is substantially free of flavokawain B and/or flavokawain A.

In certain embodiments, the composition comprises at least fourcompounds selected from the group consisting of dihydromethysticin,methysticin, dihydrokavain, kavain and desmethoxyyangonin, wherein thecomposition is substantially free of flavokawain B and/or flavokawain A.

In certain embodiments, the composition comprises 11-methoxyyangonin.

In certain embodiments, the composition comprises flavanone:

In certain embodiments, the composition comprises 11-methoxyyangonin andflavanone:

In certain embodiments, the composition is substantially free offlavokawain B.

In certain embodiments, the composition is substantially free offlavokawain A.

In certain embodiments, the composition is substantially free of bornylester of 3,4-methylenedioxy cinnamic acid:

In certain embodiments, the composition is substantially free of bornylester of cinnamic acid:

In certain embodiments, the composition is substantially free offlavanone:

In certain embodiments, the composition is substantially free ofpinostrobin.

In certain embodiments, the composition is substantially free ofmethysticin.

Certain embodiments of the invention provide a composition comprising atleast one compound selected from the group consisting ofdihydromethysticin, wherein the weight percent of dihydromethysticin inthe composition is 15±5%; methysticin, wherein the weight percent ofmethysticin in the composition is 6±5%; dihydrokavain, wherein theweight percent of dihydrokavain in the composition is 30±5%; kavain,wherein the weight percent of kavain in the composition is 29±5%;desmethoxyyangonin wherein the weight percent of desmethoxyyangonin inthe composition is 12±5%; and 11-methoxyyangonin wherein the weightpercent of 11-methoxyyangonin in the composition is 0.8±0.5%.

In certain embodiments, the at least one compound is dihydromethysticin,and wherein the weight percent of dihydromethysticin in the compositionis 15±5%.

In certain embodiments, the at least one compound is kavain, and whereinthe weight percent of kavain in the composition is 29±5%.

In certain embodiments, the at least one compound is methysticin, andwherein the weight percent of methysticin in the composition is 6±5%.

In certain embodiments, the at least one compound is dihydrokavain, andwherein the weight percent of dihydrokavain in the composition is 30±5%.

In certain embodiments, the at least one compound is desmethoxyyangonin,and wherein the weight percent of desmethoxyyangonin in the compositionis 12±5%.

In certain embodiments, the at least one compound is 11-methoxyyangonin,and wherein the weight percent of 11-methoxyyangonin in the compositionis 0.8±0.5%.

In certain embodiments of the invention, the composition comprises atleast two compounds selected from the group consisting ofdihydromethysticin, wherein the weight percent of dihydromethysticin inthe composition is 15±5%; methysticin, wherein the weight percent ofmethysticin in the composition is 6±5%; dihydrokavain, wherein theweight percent of dihydrokavain in the composition is 30±5%; kavain,wherein the weight percent of kavain in the composition is 29±5%;desmethoxyyangonin wherein the weight percent of desmethoxyyangonin inthe composition is 12±5%; and 11-methoxyyangonin wherein the weightpercent of 11-methoxyyangonin in the composition is 0.8±0.5%.

In certain embodiments of the invention, the composition comprises atleast three compounds selected from the group consisting ofdihydromethysticin, wherein the weight percent of dihydromethysticin inthe composition is 15±5%; methysticin, wherein the weight percent ofmethysticin in the composition is 6±5%; dihydrokavain, wherein theweight percent of dihydrokavain in the composition is 30±5%; kavain,wherein the weight percent of kavain in the composition is 29±5%;desmethoxyyangonin wherein the weight percent of desmethoxyyangonin inthe composition is 12±5%; and 11-methoxyyangonin wherein the weightpercent of 11-methoxyyangonin in the composition is 0.8±0.5%.

In certain embodiments of the invention, the composition comprises atleast four compounds selected from the group consisting ofdihydromethysticin, wherein the weight percent of dihydromethysticin inthe composition is 15±5%; methysticin, wherein the weight percent ofmethysticin in the composition is 6±5%; dihydrokavain, wherein theweight percent of dihydrokavain in the composition is 30±5%; kavain,wherein the weight percent of kavain in the composition is 29±5%;desmethoxyyangonin wherein the weight percent of desmethoxyyangonin inthe composition is 12±5%; and 11-methoxyyangonin wherein the weightpercent of 11-methoxyyangonin in the composition is 0.8±0.5%.

In certain embodiments of the invention, the composition comprises atleast five compounds selected from the group consisting ofdihydromethysticin, wherein the weight percent of dihydromethysticin inthe composition is 15±5%; methysticin, wherein the weight percent ofmethysticin in the composition is 6±5%; dihydrokavain, wherein theweight percent of dihydrokavain in the composition is 30±5%; kavain,wherein the weight percent of kavain in the composition is 29±5%;desmethoxyyangonin wherein the weight percent of desmethoxyyangonin inthe composition is 12±5%; and 11-methoxyyangonin wherein the weightpercent of 11-methoxyyangonin in the composition is 0.8±0.5%.

In certain embodiments of the invention, the composition comprisesdihydromethysticin, wherein the weight percent of dihydromethysticin inthe composition is 15±5%; methysticin, wherein the weight percent ofmethysticin in the composition is 6±5%; dihydrokavain, wherein theweight percent of dihydrokavain in the composition is 30±5%; kavain,wherein the weight percent of kavain in the composition is 29±5%;desmethoxyyangonin wherein the weight percent of desmethoxyyangonin inthe composition is 12±5%; and 11-methoxyyangonin wherein the weightpercent of 11-methoxyyangonin in the composition is 0.8±0.5%.

In certain embodiments, the composition further comprises flavanone:

and

wherein the weight percent of the flavanone in the composition is1±0.5%.

In certain embodiments, the composition is substantially free offlavokawain B.

In certain embodiments, the composition is substantially free offlavokawain A.

In certain embodiments, the composition is substantially free of bornylester of 3,4-methylenedioxy cinnamic acid:

In certain embodiments, the composition is substantially free of bornylester of cinnamic acid:

In certain embodiments, the composition is substantially free offlavanone:

In certain embodiments, the composition is substantially free ofpinostrobin.

Certain embodiments of the invention provide a composition comprising atleast one compound selected from the group consisting ofdihydromethysticin, wherein the weight percent of dihydromethysticin inthe composition is about 20 to 99%; methysticin, wherein the weightpercent of methysticin in the composition is about 10 to 99%;dihydrokavain, wherein the weight percent of dihydrokavain in thecomposition is about 40 to 99%; kavain, wherein the weight percent ofkavain in the composition is about 40 to 99%; desmethoxyyangonin,wherein the weight percent of desmethoxyyangonin in the composition isabout 30 to 99%; and 11-methoxyyangonin, wherein the weight percent of11-methoxyyangonin in the composition is about 20 to 99%.

In certain embodiments, the at least one compound is dihydromethysticin,wherein the weight percent of dihydromethysticin in the composition isabout 20 to 99% (e.g., about 25 to 95%, about 30 to 90%, about 35 to85%, about 40 to 80%, about 45 to 75%, about 50 to 70%, or about 55 to65%).

In certain embodiments, the at least one compound is kavain, wherein theweight percent of kavain in the composition is about 40 to 99% (e.g.,about 45 to 95%, about 50 to 90%, about 55 to 85%, about 60 to 80%, orabout 65 to 75%).

In certain embodiments, the at least one compound is methysticin,wherein the weight percent of methysticin in the composition is about 10to 99% (e.g., about 15 to 95%, about 20 to 90%, about 25 to 85%, about30 to 80%, about 35 to 75%, about 40 to 70%, about 45 to 65%, or about50 to 60%).

In certain embodiments, the at least one compound is dihydrokavain,wherein the weight percent of dihydrokavain in the composition is about40 to 99% (e.g., about 45 to 95%, about 50 to 90%, about 55 to 85%,about 60 to 80%, or about 65 to 75%).

In certain embodiments, the at least one compound is desmethoxyyangonin,wherein the weight percent of desmethoxyyangonin in the composition is30 to 99% (e.g., about 35 to 95%, about 40 to 90%, about 45 to 85%,about 50 to 80%, about 55 to 75%, or about 60 to 70%).

In certain embodiments, the at least one compound is 11-methoxyyangonin,wherein the weight percent of 11-methoxyyangonin in the composition is20 to 99% (e.g., about 25 to 95%, about 30 to 90%, about 35 to 85%,about 40 to 80%, about 45 to 75%, about 50 to 70%, or about 55 to 65%).

In certain embodiments, the composition is substantially free ofmethysticin.

In certain embodiments, the composition further comprises flavanone:

and

wherein the weight percent of the flavanone in the composition is about20 to 99% (e.g., about 25 to 95%, about 30 to 90%, about 35 to 85%,about 40 to 80%, about 45 to 75%, about 50 to 70%, or about 55 to 65%).

In certain embodiments, the composition is substantially free offlavokawain B.

In certain embodiments, the composition is substantially free offlavokawain A.

In certain embodiments, the composition is substantially free of bornylester of 3,4-methylenedioxy cinnamic acid:

In certain embodiments, the composition is substantially free of bornylester of cinnamic acid:

In certain embodiments, the composition is substantially free offlavanone:

In certain embodiments, the composition is substantially free ofpinostrobin.

In certain embodiments, the composition is a kava extract.

Certain embodiments of the invention provide a kava extract comprisingat least one compound selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin, wherein the extract issubstantially free of flavokawain B and/or flavokawain A.

In certain embodiments, the at least one compound is dihydrokavain.

In certain embodiments, the at least one compound is kavain.

In certain embodiments, the at least one compound is methysticin.

In certain embodiments, the at least one compound is dihydromethysticin.

In certain embodiments, the at least one compound is desmethoxyyangonin.

In certain embodiments, the at least one compound is 11-methoxyyangonin.

In certain embodiments of the invention, the kava extract comprises atleast two compounds selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin, wherein the extract issubstantially free of flavokawain B and/or flavokawain A.

In certain embodiments of the invention, the kava extract comprises atleast three compounds selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin, wherein the extract issubstantially free of flavokawain B and/or flavokawain A.

In certain embodiments of the invention, the kava extract comprises atleast four compounds selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin, wherein the extract issubstantially free of flavokawain B and/or flavokawain A.

In certain embodiments of the invention, the kava extract comprises atleast five compounds selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin, wherein the extract issubstantially free of flavokawain B and/or flavokawain A.

In certain embodiments of the invention, the kava extract comprisesdihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin, wherein the extract issubstantially free of flavokawain B and/or flavokawain A.

In certain embodiments, the kava extract further comprises flavanone:

In certain embodiments, the kava extract is substantially free offlavokawain B.

In certain embodiments, the kava extract is substantially free offlavokawain A.

In certain embodiments, the kava extract is substantially free of bornylester of 3,4-methylenedioxy cinnamic acid:

In certain embodiments, the kava extract is substantially free of bornylester of cinnamic acid:

In certain embodiments, the kava extract is substantially free offlavanone:

In certain embodiments, the kava extract is substantially free ofpinostrobin.

Certain embodiments of the invention provide a kava extract comprising11-methoxyyangonin and/or flavanone:

and at least one compound selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain anddesmethoxyyangonin, wherein the extract is substantially free offlavokawain B and/or flavokawain A.

In certain embodiments, the at least one compound is dihydrokavain.

In certain embodiments, the at least one compound is kavain.

In certain embodiments, the at least one compound is methysticin.

In certain embodiments, the at least one compound is dihydromethysticin.

In certain embodiments, the at least one compound is desmethoxyyangonin.

In certain embodiments, the kava extract is substantially free ofmethysticin.

In certain embodiments, the kava extract is substantially free offlavokawain B.

In certain embodiments, the kava extract is substantially free offlavokawain A.

In certain embodiments, the kava extract is substantially free of bornylester of 3,4-methylenedioxy cinnamic acid:

In certain embodiments, the kava extract is substantially free of bornylester of cinnamic acid:

In certain embodiments, the kava extract is substantially free offlavanone:

In certain embodiments, the kava extract is substantially free ofpinostrobin.

Certain embodiments of the invention provide a kava extract consistingessentially of dihydromethysticin, 11-methoxyyangonin,desmethoxyyangonin, dihydrokavain, kavain, methysticin and flavanone:

Certain embodiments of the invention provide a kava extract consistingessentially of dihydromethysticin, 11-methoxyyangonin,desmethoxyyangonin, dihydrokavain, kavain and methysticin.

Certain embodiments of the invention provide a kava extract consistingessentially of dihydromethysticin, dihydrokavain, kavain andmethysticin.

Certain embodiments of the invention provide a composition or kavaextract as described herein, wherein the composition or extract issuitable for ingestion by a mammal (e.g., a human or a dog).

Certain embodiments of the invention provide a composition or kavaextract formulated in a tablet, capsule, powder, spray, chewing gum,inhalable, patch, nano-emulsion, cream, gel, stent or liquid.

Certain embodiments of the invention provide a pharmaceuticalcomposition comprising a composition or kava extract as described hereinand a pharmaceutically acceptable carrier.

Certain embodiments of the invention provide a method, comprising:

-   -   a) combining an ethanolic kava extract and silica gel to provide        a mixture;    -   b) evaporating the mixture to provide a silica gel having kava        residue adsorbed thereon;    -   c) loading the silica gel having kava residue adsorbed thereon        on a chromatography column to provide a kava-adsorbed silica gel        column;    -   d) eluting the kava-adsorbed silica gel column with a solvent        system to provide a first kava extract fraction, a second kava        extract fraction, and a third kava extract fraction, wherein the        first kava extract fraction consists essentially of non-polar        compounds, including flavokawains, the second kava extract        fraction consists essentially of kavalactones and flavanones,        and the third kava extract fraction consists essentially of        polar compounds.

In certain embodiments, step d) comprises eluting with 28% ethyl acetate(EA) and 72% hexane (Hex) 5 column volumes (CV), followed by 90% EA and10% Hex 4.1 CV, and then 35% MeOH and 65% EA 5.5 CV.

In certain embodiments, the weight ratio of kava residue to the totalweight of silica gel having kava residue adsorbed thereon is 0.3±0.2.

In certain embodiments, the weight ratio of kava residue to the totalweight of silica gel having kava residue adsorbed thereon is 0.3±0.15.

In certain embodiments, the weight ratio of kava residue to the totalweight of silica gel having kava residue adsorbed thereon is 0.3±0.1.

Certain embodiments of the invention provide a second kava extractfraction prepared by a method described herein.

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal (e.g., a human or a dog) in need of suchtreatment comprising, administering to the mammal a carrier and acompound selected from the group consisting of dihydromethysticin,methysticin, dihydrokavain, kavain, desmethoxyyangonin and11-methoxyyangonin, wherein the compound is substantially free of otherkava extract components.

Certain embodiments of the invention provide a composition comprising acarrier and a compound selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin, for use in medical therapy,wherein the compound is substantially free of other kava extractcomponents.

Certain embodiments of the invention provide a composition comprising acarrier and a compound selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin, for the prophylactic ortherapeutic treatment of cancer, wherein the compound is substantiallyfree of other kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising a carrier and a compound selected from the group consistingof dihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin, to prepare a medicament forpreventing or treating cancer in a mammal, wherein the compound issubstantially free of other kava extract components.

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal (e.g., a human or a dog) in need of suchtreatment comprising, administering to the mammal dihydromethysticin anda carrier, wherein the dihydromethysticin is substantially free of otherkava extract components.

Certain embodiments of the invention provide a composition comprisingdihydromethysticin and a carrier for the prophylactic or therapeutictreatment of cancer in a mammal, wherein the dihydromethysticin issubstantially free of other kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising dihydromethysticin and a carrier to prepare a medicament fortreating or preventing cancer in a mammal, wherein thedihydromethysticin is substantially free of other kava extractcomponents.

Certain embodiments of the invention provide the use of a compositioncomprising dihydromethysticin and a carrier for use in medical therapy,wherein the dihydromethysticin is substantially free of other kavaextract components.

In certain embodiments, the dihydromethysticin is(+)-dihydromethysticin.

In certain embodiments, the dihydromethysticin is(±)-dihydromethysticin.

In certain embodiments, the dihydromethysticin is enriched in(+)-dihydromethysticin (e.g., at least about 51%, 60%, 70%, 80%, 90%,95% or 99% (+)-dihydromethysticin).

In certain embodiments, the dihydromethysticin is enriched in(−)-dihydromethysticin (e.g., at least about 51%, 60%, 70%, 80%, 90%,95% or 99% (−)-dihydromethysticin).

In certain embodiments, the dihydromethysticin is(−)-dihydromethysticin.

In certain embodiments, the other kava extract components are selectedfrom the group consisting of 11-methoxyyangonin, desmethoxyyangonin,dihydrokavain, kavain, methysticin, pinostrobin, flavokawain B,flavokawain A,

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal in need of such treatment comprising,administering to the mammal methysticin and a carrier, wherein themethysticin is substantially free of other kava extract components.

Certain embodiments of the invention provide a composition comprisingmethysticin and a carrier for the prophylactic or therapeutic treatmentof cancer in a mammal, wherein the methysticin is substantially free ofother kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising methysticin and a carrier to prepare a medicament fortreating or preventing cancer in a mammal, wherein the methysticin issubstantially free of other kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising methysticin and a carrier for use in medical therapy, whereinthe methysticin is substantially free of other kava extract components.

In certain embodiments, the other kava extract components are selectedfrom the group consisting of 11-methoxyyangonin, desmethoxyyangonin,dihydrokavain, kavain, dihydromethysticin, pinostrobin, flavokawain B,flavokawain A,

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal in need of such treatment comprising,administering to the mammal dihydrokavain and a carrier, wherein thedihydrokavain is substantially free of other kava extract components.

Certain embodiments of the invention provide a composition comprisingdihydrokavain and a carrier for the prophylactic or therapeutictreatment of cancer in a mammal, wherein the dihydrokavain issubstantially free of other kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising dihydrokavain and a carrier to prepare a medicament fortreating or preventing cancer in a mammal, wherein the dihydrokavain issubstantially free of other kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising dihydrokavain and a carrier for use in medical therapy,wherein the dihydrokavain is substantially free of other kava extractcomponents.

In certain embodiments, the other kava extract components are selectedfrom the group consisting of 11-methoxyyangonin, desmethoxyyangonin,dihydromethysticin, kavain, methysticin, pinostrobin, flavokawain B,flavokawain A,

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal in need of such treatment comprising,administering to the mammal kavain and a carrier, wherein the kavain issubstantially free of other kava extract components.

Certain embodiments of the invention provide a composition comprisingkavain and a carrier for the prophylactic or therapeutic treatment ofcancer in a mammal, wherein the kavain is substantially free of otherkava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising kavain and a carrier to prepare a medicament for treating orpreventing cancer in a mammal, wherein the kavain is substantially freeof other kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising kavain and a carrier for use in medical therapy, wherein thekavain is substantially free of other kava extract components.

In certain embodiments, the other kava extract components are selectedfrom the group consisting of 11-methoxyyangonin, desmethoxyyangonin,dihydrokavain, dihydromethysticin, methysticin, pinostrobin, flavokawainB, flavokawain A,

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal in need of such treatment comprising,administering to the mammal desmethoxyyangonin and a carrier, whereinthe desmethoxyyangonin is substantially free of other kava extractcomponents.

Certain embodiments of the invention provide a composition comprisingdesmethoxyyangonin and a carrier for the prophylactic or therapeutictreatment of cancer in a mammal, wherein the desmethoxyyangonin issubstantially free of other kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising desmethoxyyangonin and a carrier to prepare a medicament fortreating or preventing cancer in a mammal, wherein thedesmethoxyyangonin is substantially free of other kava extractcomponents.

Certain embodiments of the invention provide the use of a compositioncomprising desmethoxyyangonin and a carrier for use in medical therapy,wherein the desmethoxyyangonin is substantially free of other kavaextract components.

In certain embodiments, the other kava extract components are selectedfrom the group consisting of 11-methoxyyangonin, dihydromethysticin,dihydrokavain, kavain, methysticin, pinostrobin, flavokawain B,flavokawain A,

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal in need of such treatment comprising,administering to the mammal 11-methoxyyangonin and a carrier, whereinthe 11-methoxyyangonin is substantially free of other kava extractcomponents.

Certain embodiments of the invention provide a composition comprising11-methoxyyangonin and a carrier for the prophylactic or therapeutictreatment of cancer in a mammal, wherein the 11-methoxyyangonin issubstantially free of other kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising 11-methoxyyangonin and a carrier to prepare a medicament fortreating or preventing cancer in a mammal, wherein the11-methoxyyangonin is substantially free of other kava extractcomponents.

Certain embodiments of the invention provide the use of a compositioncomprising 11-methoxyyangonin and a carrier for use in medical therapy,wherein the 11-methoxyyangonin is substantially free of other kavaextract components.

In certain embodiments, the other kava extract components are selectedfrom the group consisting of desmethoxyyangonin, dihydromethysticin,dihydrokavain, kavain, methysticin, pinostrobin, flavokawain B,flavokawain A,

In certain embodiments, the other kava extract components are selectedfrom the group consisting of pinostrobin, flavokawain B, flavokawain A,

Certain embodiments of the invention provide a method for treating orpreventing cancer in a mammal in need of such treatment comprising,administering to the mammal a composition or kava extract as describedherein.

Certain embodiments of the invention provide, a composition or kavaextract as described herein for the prophylactic or therapeutictreatment of cancer in a mammal.

Certain embodiments of the invention provide the use of a composition orkava extract as described herein to prepare a medicament for treating orpreventing cancer in a mammal.

Certain embodiments of the invention provide a composition or kavaextract as described herein for use in medical therapy.

In certain embodiments, the cancer is lung cancer, prostate cancer, skincancer, melanoma, genitourinary cancer, colon and rectum cancer, breastcancer, ovarian cancer, esophagial cancer, pancreatic cancer, urinarybladder cancer, cervical cancer, liver cancer, kidney and renal cancer,head and neck cancer, brain cancer or various hematological cancers.

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal a carrier and acompound selected from the group consisting of dihydromethysticin,methysticin, dihydrokavain, kavain, desmethoxyyangonin and11-methoxyyangonin, wherein the compound is substantially free of otherkava extract components.

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal a composition or kavaextract as described herein.

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage, and/ordetoxifying physical or chemical carcinogens in a mammal (e.g., a human)in need of such treatment comprising, administering to the mammaldihydromethysticin and a carrier, wherein the dihydromethysticin issubstantially free of other kava extract components.

Certain embodiments of the invention provide a composition comprisingdihydromethysticin and a carrier for preventing tumorigenesis, reducingDNA damage, reducing protein damage, and/or detoxifying physical orchemical carcinogens in a mammal, wherein the dihydromethysticin issubstantially free of other kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising dihydromethysticin and a carrier to prepare a medicament forpreventing tumorigenesis, reducing DNA damage, reducing protein damage,and/or detoxifying physical or chemical carcinogens in a mammal, whereinthe dihydromethysticin is substantially free of other kava extractcomponents.

In certain embodiments, the dihydromethysticin is(+)-dihydromethysticin.

In certain embodiments, the dihydromethysticin is(f)-dihydromethysticin.

In certain embodiments, the dihydromethysticin is enriched in(+)-dihydromethysticin (e.g., at least about 51%, 60%, 70%, 80%, 90%,95% or 99% (+)-dihydromethysticin).

In certain embodiments, the dihydromethysticin is enriched in(−)-dihydromethysticin (e.g., at least about 51%, 60%, 70%, 80%, 90%,95% or 99% (−)-dihydromethysticin).

In certain embodiments, the dihydromethysticin is(−)-dihydromethysticin.

In certain embodiments, the other kava extract components are selectedfrom the group consisting of 11-methoxyyangonin, desmethoxyyangonin,dihydrokavain, kavain, methysticin, pinostrobin, flavokawain B,flavokawain A,

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage, and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal methysticin and acarrier, wherein the methysticin is substantially free of other kavaextract components.

Certain embodiments of the invention provide a composition comprisingmethysticin and a carrier for preventing tumorigenesis, reducing DNAdamage, reducing protein damage, and/or detoxifying physical or chemicalcarcinogens in a mammal, wherein the methysticin is substantially freeof other kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising methysticin and a carrier to prepare a medicament forpreventing tumorigenesis, reducing DNA damage, reducing protein damage,and/or detoxifying physical or chemical carcinogens in a mammal, whereinthe methysticin is substantially free of other kava extract components.

In certain embodiments, the other kava extract components are selectedfrom the group consisting of 11-methoxyyangonin, desmethoxyyangonin,dihydrokavain, kavain, dihydromethysticin, pinostrobin, flavokawain B,flavokawain A,

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage, and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal dihydrokavain and acarrier, wherein the dihydrokavain is substantially free of other kavaextract components.

Certain embodiments of the invention provide a composition comprisingdihydrokavain and a carrier for preventing tumorigenesis, reducing DNAdamage, reducing protein damage, and/or detoxifying physical or chemicalcarcinogens in a mammal, wherein the dihydrokavain is substantially freeof other kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising dihydrokavain and a carrier to prepare a medicament forpreventing tumorigenesis, reducing DNA damage, reducing protein damage,and/or detoxifying physical or chemical carcinogens in a mammal, whereinthe dihydrokavain is substantially free of other kava extractcomponents.

In certain embodiments, the other kava extract components are selectedfrom the group consisting of 11-methoxyyangonin, desmethoxyyangonin,dihydromethysticin, kavain, methysticin, pinostrobin, flavokawain B,flavokawain A,

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal kavain and a carrier,wherein the kavain is substantially free of other kava extractcomponents.

Certain embodiments of the invention provide a composition comprisingkavain and a carrier for preventing tumorigenesis, reducing DNA damage,reducing protein damage and/or detoxifying physical or chemicalcarcinogens in a mammal, wherein the kavain is substantially free ofother kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising kavain and a carrier to prepare a medicament for preventingtumorigenesis, reducing DNA damage, reducing protein damage, and/ordetoxifying physical or chemical carcinogens in a mammal, wherein thekavain is substantially free of other kava extract components.

In certain embodiments, the other kava extract components are selectedfrom the group consisting of 11-methoxyyangonin, desmethoxyyangonin,dihydrokavain, dihydromethysticin, methysticin, pinostrobin, flavokawainB, flavokawain A,

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal desmethoxyyangonin anda carrier, wherein the desmethoxyyangonin is substantially free of otherkava extract components.

Certain embodiments of the invention provide a composition comprisingdesmethoxyyangonin and a carrier for preventing tumorigenesis, reducingDNA damage, reducing protein damage and/or detoxifying physical orchemical carcinogens in a mammal, wherein the desmethoxyyangonin issubstantially free of other kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising desmethoxyyangonin and a carrier to prepare a medicament forpreventing tumorigenesis, reducing DNA damage, reducing protein damageand/or detoxifying physical or chemical carcinogens in a mammal, whereinthe desmethoxyyangonin is substantially free of other kava extractcomponents.

In certain embodiments, the other kava extract components are selectedfrom the group consisting of 11-methoxyyangonin, kavain, dihydrokavain,dihydromethysticin, methysticin, pinostrobin, flavokawain B, flavokawainA,

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal 11-methoxyyangonin anda carrier, wherein the 11-methoxyyangonin is substantially free of otherkava extract components.

Certain embodiments of the invention provide a composition comprising11-methoxyyangonin and a carrier for preventing tumorigenesis, reducingDNA damage, reducing protein damage and/or detoxifying physical orchemical carcinogens in a mammal, wherein the 11-methoxyyangonin issubstantially free of other kava extract components.

Certain embodiments of the invention provide the use of a compositioncomprising 11-methoxyyangonin and a carrier to prepare a medicament forpreventing tumorigenesis, reducing DNA damage, reducing protein damageand/or detoxifying physical or chemical carcinogens in a mammal, whereinthe 11-methoxyyangonin is substantially free of other kava extractcomponents.

In certain embodiments, the other kava extract components are selectedfrom the group consisting of desmethoxyyangonin, kavain, dihydrokavain,dihydromethysticin, methysticin, pinostrobin, flavokawain B, flavokawainA,

In certain embodiments, the other kava extract components are selectedfrom the group consisting of pinostrobin, flavokawain B, flavokawain A,

Certain embodiments of the invention provide a method for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal in need of suchtreatment comprising, administering to the mammal a composition or kavaextract as described herein.

Certain embodiments of the invention provide a composition or kavaextract as described herein for preventing tumorigenesis, reducing DNAdamage, reducing protein damage and/or detoxifying physical or chemicalcarcinogens in a mammal.

Certain embodiments of the invention provide the use of a composition orkava extract as described herein to prepare a medicament for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal.

In certain embodiments, the DNA damage is reduced by about 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%, as compared to a mammal not administered acomposition or kava extract as described herein.

In certain embodiments, the protein damage is reduced by about 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100%, as compared to a mammal not administered acomposition or kava extract as described herein.

In certain embodiments, the DNA damage is a DNA adduct, caused byphysical or chemical carcinogens.

In certain embodiments, the DNA adduct is a O⁶-methylguanine DNA adduct.

In certain embodiments, the DNA adduct is a 7-methylguanine DNA adduct.

In certain embodiments, the DNA adducts are BaP, PhIP, POB and/or PHBadducts. In certain embodiments, the DNA adducts are POB and PHB DNAadducts (e.g., 7-pobG, 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine; O²-pobdT,O²-[4-(3-pyridyl)-4-oxobut-1yl]thymidine; O⁶-pobdG,O⁶-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine; O²-pobC,O²-[4-(3-pyridyl)-4-oxobut-1-yl]cytidine).

Certain embodiments of the invention provide a composition comprising acarrier and a compound selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin for use in medical therapy,wherein the compound is substantially free of other kava extractcomponents.

Certain embodiments of the invention provide a composition comprising acarrier and a compound selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin for the prophylactic ortherapeutic treatment of cancer, wherein the compound is substantiallyfree of other kava extract components.

Certain embodiments of the invention provide a composition comprising acarrier and a compound selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin to prepare a medicament forpreventing or treating cancer in a mammal, wherein the compound issubstantially free of other kava extract components.

In certain embodiments, the compound is dihydromethysticin.

In certain embodiments, the compound is methysticin.

In certain embodiments, the compound is dihydrokavain.

In certain embodiments, the compound is kavain.

In certain embodiments, the compound is desmethoxyyangonin.

In certain embodiments, the compound is 11-methoxyyangonin.

The invention also provides processes and intermediates disclosed hereinthat are useful for preparing compositions and extracts described herein(see, e.g., the Examples).

As used herein, the phrase “substantially free of” means the compositionor kava extract comprises less than about 5%, 4%, 3%, 2%, 1%, 0.9%,0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or 0.05% by weight of thedesignated compound(s) and/or components.

As used herein, the phrase “consisting essentially of” means thecomposition or kava extract comprises less than about 5%, 4%, 3%, 2%,1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or 0.05% byweight of other compounds and/or components.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) an undesired physiological change ordisorder, such as the growth, development or spread of cancer. Forpurposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, alleviation of symptoms, diminishmentof extent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented. Asdescribed in the Examples, the compositions, kava extracts and compounds(e.g., such as, dihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin) described herein, havechemopreventive properties, and therefore, are useful for both thetreatment and prevention of cancer.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell accumulation. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.Specific examples of cancers include, but are not limited to, lungcancer, prostate cancer, skin cancer, melanoma, genitourinary cancer,colon and rectum cancer, breast cancer, ovary cancer, esophagial cancer,pancreatic cancer, urinary bladder cancer, cervical cancer, livercancer, kidney and renal cancer, head and neck cancer, brain cancer orvarious hematological cancers. Examples of cancer also include, but arenot limited to, cancerous lesions in the above tissues, such as familialadenomatous polyposis, hyperplasia, dysplasia, aberrant crypt foci,adenoma, and others.

The phrase “detoxifying physical or chemical carcinogens” refers toenhancing elimination/deactivation of toxic species generated fromphysical or chemical carcinogens, reducing the generation of toxicspecies from physical or chemical carcinogens, and/or activating theimmune system to improve self-defense against physical or chemicalcarcinogens.

As used herein, the terms “protein damage” refers to natural proteins,such as hemoglobin, being modified by the reactive metabolites/speciesgenerated by the chemical or physical carcinogens (Murphy et al.,Chemico-Biological Interactions 1997; 103:153-166). Methods formeasuring protein damage are known in the art, for example, as describedin Murphy et al., Chemico-Biological Interactions 1997; 103:153-166.

As used herein, the term “mammal” includes, but is not limited to,humans, mice, rats, guinea pigs, monkeys, dogs, cats, horses, cows,pigs, and sheep, and poultry.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase).

When a bond in a compound formula herein is drawn in anon-stereochemical manner (e.g. flat), the atom to which the bond isattached includes all stereochemical possibilities. For example, incertain embodiments, all stereochemical possibilities are included forthe following compounds:

When a bond in a compound formula herein is drawn in a definedstereochemical manner (e.g. bold, bold-wedge, dashed or dashed-wedge),it is to be understood that the atom to which the stereochemical bond isattached is enriched in the absolute stereoisomer depicted unlessotherwise noted. In one embodiment, the compound may be at least 51% theabsolute stereoisomer depicted. In another embodiment, the compound maybe at least 60% the absolute stereoisomer depicted. In anotherembodiment, the compound may be at least 80% the absolute stereoisomerdepicted. In another embodiment, the compound may be at least 90% theabsolute stereoisomer depicted. In another embodiment, the compound maybe at least 95 the absolute stereoisomer depicted. In anotherembodiment, the compound may be at least 99% the absolute stereoisomerdepicted. For example, in certain embodiments, the atom to which thestereochemical bond is attached is enriched in the absolute stereoisomerdepicted for the following compounds:

In certain embodiments, the invention provides a composition enriched innon-natural (−)-dihyromethysticin:

The compositions, kava extracts and compounds described herein (e.g.,such as, dihydromethysticin, methysticin, dihydrokavain, kavain,desmethoxyyangonin and 11-methoxyyangonin) can be formulated ascompositions (e.g., pharmaceutical) and administered to a mammalianhost, such as a human patient in a variety of forms adapted to thechosen route of administration, i.e., orally or parenterally, byintravenous, intramuscular, topical or subcutaneous routes.

Thus, the present compositions, kava extracts and compounds may besystemically administered, e.g., orally, in combination with apharmaceutically acceptable vehicle such as an inert diluent or anassimilable edible carrier. They may be enclosed in hard or soft shellgelatin capsules, may be compressed into tablets, or may be incorporateddirectly with the food and drinks of the patient's diet. For oraltherapeutic administration, the present compositions, kava extracts andcompounds may be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, spray, nano-emulsion, patch, cream, gum, gel,stent, wafers, drinks and the like. Such compositions and preparationsshould contain at least 0.01% of the present compositions, kava extractsand compounds. The percentage of the compositions and preparations may,of course, be varied and may conveniently be between about 0.1 to about60% of the weight of a given unit dosage form. The amount of the presentcompositions, kava extracts and compounds in such therapeutically usefulcompositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the present compositions, kava extracts or compounds, sucrose orfructose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any unit dosage form should bepharmaceutically acceptable and substantially non-toxic in the amountsemployed. In addition, the present compositions, kava extracts andcompounds may be incorporated into sustained-release preparations anddevices.

The present compositions, kava extracts and compounds may also beadministered intravenously or intraperitoneally by infusion orinjection. Solutions of the present compositions, kava extracts andcompounds can be prepared in water, optionally mixed with a nontoxicsurfactant. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, triacetin, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the present compositions, kava extracts and compounds whichare adapted for the extemporaneous preparation of sterile injectable orinfusible solutions or dispersions, optionally encapsulated inliposomes. In all cases, the ultimate dosage form should be sterile,fluid and stable under the conditions of manufacture and storage. Theliquid carrier or vehicle can be a solvent or liquid dispersion mediumcomprising, for example, water, ethanol, a polyol (for example,glycerol, propylene glycol, liquid polyethylene glycols, and the like),vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.The proper fluidity can be maintained, for example, by the formation ofliposomes, by the maintenance of the required particle size in the caseof dispersions or by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, buffers orsodium chloride. Prolonged absorption of the injectable compositions canbe brought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the presentcompositions, kava extracts or compounds in the required amount in theappropriate solvent with various of the other ingredients enumeratedabove, as required, followed by filter sterilization. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze dryingtechniques, which yield a powder of the present compositions, kavaextracts and compounds plus any additional desired ingredient present inthe previously sterile-filtered solutions.

For topical administration, the present compositions, kava extracts andcompounds may be applied in pure form, i.e., when they are liquids.However, it will generally be desirable to administer them to the skinas compositions or formulations, in combination with a dermatologicallyacceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compositions, kava extracts and compoundscan be dissolved or dispersed at effective levels, optionally with theaid of non-toxic surfactants. Adjuvants such as fragrances andadditional antimicrobial agents can be added to optimize the propertiesfor a given use. The resultant liquid compositions can be applied fromabsorbent pads, used to impregnate bandages and other dressings, orsprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the present compositions, kava extracts and compounds to theskin are known to the art; for example, see Jacquet et al. (U.S. Pat.No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat.No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the present compositions, kava extracts and compoundscan be determined by comparing their in vitro activity, and in vivoactivity in animal models. Methods for the extrapolation of effectivedosages in mice, and other animals, to humans are known to the art; forexample, see U.S. Pat. No. 4,938,949.

The amount of the present compositions, kava extracts or compounds,required for use in the treatment or prevention of cancer, prevention oftumorigenesis, reduction of DNA and protein damage and/or detoxificationof physical or chemical carcinogens will vary not only with theparticular form selected but also with the route of administration, thenature of the condition being treated and the age and condition of thepatient and will be ultimately at the discretion of the attendantphysician or clinician.

In general, however, a suitable dose will be in the range of from about0.05 to about 100 mg/kg, e.g., from about 1 to about 75 mg/kg of bodyweight per day, such as 3 to about 50 mg per kilogram body weight of therecipient per day, preferably in the range of 0.2 to 90 mg/kg/day, mostpreferably in the range of 1 to 15 mg/kg/day. For example, in certainembodiments, dihydromethysticin may be administered in the range ofabout 0.2 to 1.0 mg/kg/day, or from about 0.4 to about 0.8 mg/kg/day, orabout 0.6 mg/kg/day.

The present compositions, kava extracts and compounds are convenientlyformulated in unit dosage form; for example, containing 5 to 1000 mg,conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of thepresent compositions, kava extracts or compounds per unit dosage form.In one embodiment, the invention provides a composition comprising apresent composition, kava extract or compound of the inventionformulated in such a unit dosage form.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

The invention will now be illustrated by the following non-limitingExamples.

Example 1

As described herein, the tumorigenesis-stage specificity of kava, thepotential active compounds, and the underlying mechanisms in NNK-inducedlung tumorigenesis in A/J mice has now been investigated. In the firstexperiment, NNK-treated mice were given diets containing kava at a doseof 5 mg/g of diet during different periods. Kava treatments covering theinitiation stage reduced the multiplicity of lung adenomas by ˜99%. Aminimum effective dose is yet to be defined because kava at two lowerdosages (2.5 and 1.25 mg/g of diet) were equally effective as 5 mg/g ofdiet in complete inhibiting lung adenoma formation. Daily gavage of kava(one before, during, and after NNK treatment) completely blocked lungadenoma formation as well. Kavalactone-enriched Fraction B fullyrecapitulated kava's chemopreventive efficacy while kavalactone-freeFractions A and C were much less effective. Mechanistically, kava andFraction B reduced NNK-induced DNA damage in lung tissues with a uniqueand preferential reduction in O⁶-methylguanine (O⁶-mG), the highlytumorigenic DNA damage by NNK, correlating and predictive of efficacy onblocking lung adenoma formation. Taken together, these resultsdemonstrate the outstanding efficacy of kava in preventing NNK-inducedlung tumorigenesis in A/J mice with high selectivity for the initiationstage in association with the reduction of O⁶-mG adduct in DNA. Theyalso establish the knowledge basis for the identification of the activecompound(s) in kava.

Accordingly, based on these experiments described herein, the presentcompositions, kava extracts and compounds (e.g., dihydromethysticin,methysticin, dihydrokavain, kavain, desmethoxyyangonin and11-methoxyyangonin) may be useful for both the treatment and preventionof cancer. They are also expected to have high safety profiles relativeto typical cancer therapies and show superior efficacy and drugproperties (e.g., oral bioavailability).

INTRODUCTION

Lung cancer is the leading cause of malignancy-related mortality becauseof its high incidence and the lack of effective treatments. Sincetobacco usage contributes to 85-90% of its development, tobaccocessation is the most straightforward strategy for reducing lung cancerincidence and mortality. However, because of the addictive nature oftobacco, limited progress has been achieved in reducing tobacco usage.An alternative approach is to block or slow down tobaccocarcinogen-induced lung cancer development via chemoprevention (Hecht etal., Nat. Rev. Cancer 2009; 9:476-88). Although a number of compoundshave been identified as potential chemopreventive agents against lungtumorigenesis in animal models, their in vivo efficacy leaves ample roomfor improvement. Additional candidates with novel chemical structures,unique mechanisms, and better efficacy therefore need to be identified.

The A/J mice carry the pulmonary adenoma susceptibility 1 (Pas1) gene,tightly linked to the Kras oncogene (O'donnell et al., Cancer Lett 2006;241:197-202), so that they have high susceptibility to lung tumordevelopment. The A/J mice would develop lung tumor upon aging with hightumor incidence but low tumor multiplicity even without tobaccocarcinogen treatment. With appropriate tobacco carcinogen treatment, A/Jmice would develop lung tumors with 100% incidence and high multiplicityin a relatively short period of time (Hecht et al., Nat. Rev. Cancer2009; 9:476-88). The tumors induced also have morphological,histological and molecular features similar to human lungadenocarcinomas (Malkinson, Lung Cancer 2001; 32:265-79). Therefore, thetobacco carcinogen-treated A/J mouse model is the most commonly usedlung tumorigenesis model for evaluating chemopreventive agents withtumor multiplicity being the most practical endpoint.

Kava is an aqueous extract of the roots of Piper methysticum andtraditionally serves as a beverage for South Pacific islanders. Kava hadalso been used to treat anxiety (Boerner et al., Phytomedicine 2003; 10Suppl 4:38-49; Sarris J, et al., J Clin Psychopharmacol 2013;33(5):643-648), in which case it was prepared as an organic extract.Epidemiological surveillance detected very low cancer incidence rates inseveral South Pacific countries, including lung cancer (Henderson etal., Fourth symposium on epidemiology and cancer registries in thepacific basin 1984:73-81; Henderson et al., Natl. Cancer Inst. Monogr.1985; 69:73-81), and traditional kava usage may be a risk-loweringfactor (Steiner G G., Hawaii Med. J. 2000; 59:420-2). Kava contains aclass of unique chemicals, kavalactones (Rowe et al., Mini Rev Med Chem2011; 11:79-83), which have not been reported to prevent tumorigenesis.Kava, particularly the anxiolytic preparation, also containschalcone-based flavokawains, flavanones, and bornyl esters, which mayinhibit cancer development.

It has recently been demonstrated that dietary supplement of an ethanolkava extract at a dose of 10 mg/g of diet, during initiation stage orpost-initiation stage, effectively reduced lung adenomas multiplicityinduced by eight gavage treatment of a mixture of the well-known tobaccocarcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) andbenzo(a)pyrene (BaP) without adverse side effects in A/J mice (Johnsonet al., Am J Chin Med 2011; 39:727-42; Johnson et al., Cancer Prev Res(Phila) 2008; 1:430-8). Since NNK and BaP induce adenoma formation viadifferent mechanisms, the two-carcinogen model does not provide afeasible system to tackle questions regarding kava's underlyingmechanisms and responsible chemicals. The studies described herein weredesigned to address these questions by using an NNK-induced lungtumorigenesis A/J mouse model. Similar studies using the BaP-inducedlung tumorigenesis models may also be carried out.

Materials and Methods Abbreviations

NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; BaP,benzo(a)pyrene; O⁶-mG, O⁶-methylguanine; 7-pobG,7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine; O²-pobdT,O²-[4-(3-pyridyl)-4-oxobut-1 yl]thymidine; O⁶-pobdG,O⁶-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine; O²-pobC,O²-[4-(3-pyridyl)-4-oxobut-1-yl]cytidine; 7-mG, 7-methylguanine; O⁴-mT,O⁴-methylthymidine; CV, column volume; TLC, thin layer chromatography;H&E, hematoxylin and eosin; Pas1, pulmonary adenoma susceptibility 1;ANOVA, analysis of variance; PEITC, phenethyl isothiocyanate.

Chemicals, Reagents, and Animal Diets

NNK was synthesized (Hecht et al., Carcinogenesis 1983; 4:305-10). Thekava product was purchased from Gaia Herbs, Inc. (Brevard, N.C.). It isan ethanol extract of the wild crafted lateral root from Vanuatu(standardized to 150 mg/mL total kavalactones). The AIN-93 purifieddiets from Harlan Teklad (Madison, Wis.) were used herein. The AIN-93Gpowdered diet started one week before the first dose of NNK and endedone week after the second dose of NNK; thereafter, it was replaced byAIN-93M powdered diet. O⁶-methylguanine (O⁶-mG) was purchased fromMidwest Research Institute (Kansas City, Mo.). [CD₃]O⁶-mG was purchasedfrom Toronto Research Chemicals (Toronto, Ontario, Canada).7-[4-(3-Pyridyl)-4-oxobut-1-yl]guanine (7-pobG),O²-[4-(3-pyridyl)-4-oxobut-1 yl]thymidine (O²-pobdT),O⁶-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine (O⁶-pobdG), and thecorresponding [pyridine-D₄]analogues were synthetized (Lao et al., ChemRes Toxicol 2006; 19:674-82; Sturla et al., Chem Res Toxicol 2005;18:1048-55). Micrococcal nuclease and phosphodiesterase II were fromWorthington Biochemical Corporation (Lakewood, N.J.). Alkalinephosphatase was from Roche Molecular Biochemicals (Indianapolis, Ind.).

Kava Fractionation Preparation and Characterization

Previous investigation of traditional kava and kava from Gaia Herbsrevealed that the Gaia Herbs preparation contained some minor non-polarconstituents with high toxicity (Shaik et al., Bioorg Med Chem Lett2009; 19:5732-6). The fractionation protocol of kava from Gaia Herbsusing silica gel chromatography was developed and optimized, leading tothree modalities—Fractions A, B, and C. Briefly, 350 mL of ethanolickava extract was mixed with 350 g of silica gel. Solvents wereevaporated under vacuum. Based on mass balance, 100 gram kava-adsorbedsilica gel contained 28 gram kava residue. Kava-adsorbed silica gel (350g) was loaded on a sample loading chamber and separated by a BiotageSemi-preparative system. The elution method was 28% ethyl acetate (EA)and 72% hexane (Hex) 5 column volumes (CV), followed by 90% EA and 10%Hex 4.1 CV, and then 35% MeOH and 65% EA 5.5 CV. Different eluents wereanalyzed by thin layer chromatography (TLC) and the desired eluents werecombined with solvent removed to generate Fractions A, B, and C. Thequantity of each fraction was measured and the integrity of eachfraction was characterized by comparing the fingerprints of their ¹H-NMRspectra. These fractions were also characterized by HPLC in comparisonto traditional kava on a Beckman Coulter System Gold 126 solvent modulewith a 168 detector. A Clipeus C-18 column (5 μm, 250×4.6 mm) was usedfor the HPLC analyses. The flow rate used was 0.5 mL/min. The mobilephase A was water while B was acetonitrile. The time program used forthe analyses was 70% B (0-5 min), 70-95% B (5-30 min), and 95% B (30-35min) Compounds in Fractions B and C were further separated by normalphase silica gel chromatography and characterized by ¹H-NMR and massspectrometry.

Diet Preparation

Different kava modalities in the appropriate quantity were reconstitutedin absolute ethanol (50 mL) and then mixed with AIN-93 powdered diet(150 g). Absolute ethanol (50 mL) was also mixed with AIN-93 powdereddiet (150 g) for the control diet preparation. The reconstituted dietswere dried under vacuum to remove ethanol, ground to a fine powder andmixed with additional AIN-93 powdered diet to the desired dose. Theinitial dose of kava (5 mg/g of diet) was chosen based on the results ofprevious study showing that kava at this dose was well tolerated in A/Jmice while its lung cancer chemopreventive efficacy was similar to thatat a higher dose (Johnson et al., Am J Chin Med 2011; 39:727-42).

Experiments Assessing Efficacy of Different Kava Regimens on LungAdenoma Formation Induced by NNK in A/J Mice

Female A/J mice, 5-6 weeks of age from the Jackson Laboratory (BarHarbor, Me.), were handled according to animal welfare protocolsapproved by IACUC at the University of

Minnesota. Upon arrival, mice were housed in the specific pathogen-freeanimal facilities of Research Animal Resources, University of Minnesota.After one-week acclimation, mice were weighed, randomized into differentgroups and switched to AIN-93G-powdered diet, defined as Day 1. Thenumber of mice in each group was specified in the Result Section. On Day7 and Day 14, mice in the negative control groups received 0.1 mLphysiological saline solution while mice in the other groups receivedNNK (100 and 67 mg/kg respectively in 0.1 mL of physiological salinesolution) via i.p. injection. At the end of Day 21, mice were switchedto AIN-93M-powdered diet until the end of the study. Diet consumptionwas measured twice weekly and bodyweight was monitored weekly. All micewere euthanized with an overdose of carbon dioxide. The lungs werecollected and tumors on the surface of the lung were counted.

For Experiment 1, mice were fed diet supplemented with/without kava at adose of 5 mg/g of diet during specified periods (FIG. 4 and Table 1) todefine tumorigenesis-stage specificity. This study was terminated at theend of Day 119. For Experiment 2, mice were fed diet supplementedwith/without kava at a dose of 5 mg/g of diet during Day 1-Day 14(initiation stage only, Table 2). Half of the mice were terminated atthe end of Week 25 (Day 175) and the other half at the end of Week 34(Day 238). For Experiment 3, mice were given vehicle (PEG400:EtOH 9:1,200 μL) or kava in the same vehicle (20 mg/mouse/day) via daily gavagewith regimens specified in the Result Section. This study was terminatedat the end of Day 119 (Table 3). For Experiment 4, mice were fed dietsupplemented with kava at lower dosages to define dose-response patternor different kava fractions at a dose of 2.5 mg/g of diet during Day1-Day 14. This study was terminated at the end of Day 119 (Table 4).

Experiments Evaluating Effect of Kava on Acute DNA Adduct Formation byNNK in A/J Mice

After one-week acclimation, A/J mice were weighed and randomized intosixteen groups (3 mice per group) and switched to AIN-93G-powdered dietwith the date being defined as Day 1. Mice in Groups 1, 2, 4, 6, 8, 10,and 12 were given AIN-93G diet through the study. Mice in Groups 3, 5,7, 9, 11 and 13 were given AIN-93G diet supplemented with kava at a doseof 5 mg/g of diet through the study except for mice in Groups 9, 11, and13, which were switched to plain AIN-93G diet one day after NNKinjection to mimic stopping kava treatment one day after the last NNKtreatment in the long-term lung tumorigenesis studies. Mice in Groups14-16 were given AIN-93G diet supplemented with Fractions A, B, and Crespectively at a dose of 2.5 mg/g of diet through the study. On Day 7,mice in Group 1 received 0.1 mL physiological saline solution while micein the other groups received NNK (100 mg/kg in 0.1 mL physiologicalsaline solution) via i.p. injection. Mice in Groups 1, 2, and 3 wereeuthanized 4 h after NNK injection. Mice in Groups 4 and 5 wereeuthanized 8 h after NNK injection. Mice in Groups 6, 7, 14, 15, and 16were euthanized 24 h after NNK injection. Mice in Groups 8 and 9 wereeuthanized 48 h after NNK injection. Mice in Groups 10 and 11 wereeuthanized 96 h after NNK injection. Mice in Groups 12 and 13 wereeuthanized 2 weeks (336 h) after NNK injection. All mice were euthanizedwith an overdose of carbon dioxide. The lungs were harvested, snapfrozen in liquid N₂ and stored at −80° C. until DNA isolation.

Isolation and Quantification of DNA Adducts in the Lung Tissues byLC-ESI-MS/MS

DNA was isolated from the whole lung tissue of each individual mousefollowing Puregene DNA isolation protocol (Qiagen Corp, Valencia,Calif.) (Urban et al., Chem Res Toxicol 2012; 25:2167-78). 7-pobG,O²-pobdT, O⁶-pobdG, and O⁶-mG were quantified by LC-ESI-MS/MS, followingestablished protocols (Urban et al., Chem Res Toxicol 2012; 25:2167-78;Peterson et al., Cancer Res 1991; 51:5557-64).

Lung Tumor Histopathology

4-μm-thick sections made from formalin-fixed and paraffin embedded lungtissues were stained with hematoxylin and eosin (H&E).

Statistical Analyses

Data on lung adenoma multiplicity were reported as mean±SD (n=5−40).One-way analysis of variance (ANOVA) was used to compare means among NNKand NNK+kava modality groups for Experiments 1, 3, and 4. Dunnett's testwas used for comparisons of the number of tumors on the surface of thelung between NNK control and kava modality treatment groups. p-value≦0.05 was considered statistically significant. For Experiment 2,unpaired t-test was used for comparison between NNK control and kavatreatment groups. Two-sided p-value ≦0.05 was considered statisticallysignificant. Data on DNA adducts were reported as mean±SD (n=3). For thetime-course study, unpaired t-test was used for comparisons between NNKcontrol and kava treatment groups. Two-sided p-value <0.05 wasconsidered statistically significant: *p<0.05, **p<0.01, and ***p<0.001.For the 24-h time point study, one-way ANOVA was used to compare means.Dunnett's test was used for comparisons between NNK control and kavamodality treatment groups. p-value <0.05 was considered statisticallysignificant. All analyses were conducted in GraphPad Prism 4 (GraphPadSoftware, Inc. La Jolla, Calif.).

Results

Effect of Kava Treatment Schedule with Respect to NNK Exposure on LungAdenoma Formation in A/J Mice—Experiment 1

To test whether kava inhibited a specific stage of NNK-induced lungtumorigenesis, A/J mice were given two dosages of NNK (100 and 67 mg/kgof bodyweight on Day 7 and Day 14 respectively via i.p. injection).NNK-treated A/J mice were given diet supplemented with kava at a dose of5 mg/g of diet during different periods of time in reference to NNKexposure. Both the adenoma incidence (presence of one detectable surfaceadenoma) and the number of adenomas on the lung surface at the end ofDay 119 were quantified (Table 1).

As expected, A/J mice without NNK treatment had low adenoma incidence(10%) and low adenoma multiplicity (0.1±0.3 lung adenoma/mouse) whileNNK-treated A/J mice had 100% adenoma incidence and high adenomamultiplicity (17.5±4.8 lung adenoma/mouse). Kava treatment regimens thatstarted after the final NNK treatment (Groups 6-9, i.e.,post-initiation) had no effect on adenoma incidence. Such treatmentsalso had little effect on adenoma multiplicity, except for the Day15—119 regimen (Group 6), which reduced adenoma multiplicity by 24%(13.3±4.3 lung adenoma/mouse, p<0.05). On the other hand, kavatreatments that preceded and covered the NNK exposure period (Groups3-5, i.e., initiation stage) not only reduced adenoma incidence by67-87% but also reduced adenoma multiplicity by ˜99%, to a level similarto mice without NNK treatment, which is not expected with respect to itsoutstanding efficacy. None of the long-term kava treatment regimens(Groups 5, 6, 8 and 9) caused >10% reduction in bodyweight, and theshort-term treatment regimens (Groups 3, 4 and 7) did not reducebodyweight relative to NNK-treated mice (Group 2). None of the kavatreatment regimens caused significant changes in liver weight incomparison to NNK-treated mice (Group 2). These data indicated acomplete blocking effect of kava on NNK-induced initiation of lungtumorigenesis, with a modest post-initiation inhibitory efficacy.

Effect of Kava on Long-Term Lung Tumorigenesis in A/J Mice—Experiment 2

To validate the anti-initiation efficacy of the short kava treatmentduring NNK treatment period (Day 1-Day 14) and to determine whether suchinhibition would persist through later stages of tumorigenesis, the kavaand NNK treatment experiments for the initiation stage were replicatedand the tumor status at Week 25 (Day 175) and Week 34 (Day 238) wasanalyzed. As shown in Table 2, A/J mice without NNK treatment had noadenoma and NNK-treated A/J mice had 100% adenoma incidence and highadenoma multiplicity (18.1±5.1 lung adenoma/mouse) at Week 25. Kava at adose of 5 mg/g of diet given during Day 1-Day 14 reduced adenomaincidence by 73% and adenoma multiplicity by 98.5%. As expected oflonger duration for tumors to grow, A/J mice at Week 34 had higheradenoma multiplicity (26.5±7.8 lung adenoma/mouse) than those at Week25. A/J mice without NNK treatment also had higher incidence (25%) andmultiplicity (0.5±1.0 lung adenoma/mouse) of spontaneous tumors thanthose at Week 25. Kava given during Day 1-Day 14 did not reduce adenomaincidence but dramatically reduced adenoma multiplicity by 97.7%(1.1±0.6 lung adenoma/mouse). FIG. 5 shows representativephotomicrographs of sections of lung from mice without NNK treatment(FIG. 5A), mice with NNK treatment (FIG. 5B), and mice with NNK and kavatreatment (FIG. 5C), which confirmed tumor reduction in the lunginterior with kava treatment to the same magnitude as enumerated bycounting the visible lung surface lesions. This kava treatment regimencaused no changes in mouse bodyweight and liver weight relative to theNNK-control groups. The data from this experiment not only confirmed theinitiation-specific inhibitory efficacy of kava on NNK-induced lungtumorigenesis but also demonstrated the long-lasting protective natureof such a brief treatment.

Effect of Daily Gavage of Kava on Lung Adenoma Formation in A/JMice—Experiment 3

Given potential pharmacokinetic differences between kava consumption inhumans (most practical as a bolus dose through dietary supplementpill/drink) vs. that of continuous rodent food intake in the experimentsso far, an experiment to explore whether once daily gavage of kava mightbe as effective in preventing NNK-induced adenoma formation in A/J micewas carried out. The dose of kava, 20 mg/mouse/day, was chosen based onthe fact that A/J mouse consumes 3-4 g of diet/day and the kava dose indiet was 5 mg/g of diet. In one regimen, once daily kava gavage startedone day before the first NNK treatment and continued until one day afterthe second NNK treatment—Group 3 (Table 3). In the second regimen, oncedaily kava gavage started one day before the first NNK treatment, endedone day after the first NNK treatment, resumed one day before the secondNNK treatment and ended one day after the second NNK treatment—Group 4(Table 3). When the incidence and number of adenoma on the lung surfaceat the end of Day 119 were quantified (Table 3), A/J mice without NNKtreatment had low adenoma incidence (20%) and low adenoma multiplicity(0.2±0.4 lung adenoma/mouse) while NNK-treated A/J mice had 100% adenomaincidence and high adenoma multiplicity (16.6±3.1 lung adenoma/mouse).Both kava gavage regimens reduced adenoma incidence (60-100%) andreduced adenoma multiplicity by ˜99%. None of these regimens causedsignificant bodyweight or liver weight change in comparison to mice inGroup 2. These data therefore convincingly established the feasibilityof using kava in as few as 3 once-daily bolus treatments (i.e., one dosebefore, during and after the NNK injection) to block NNK-induced adenomainitiation. Similar experiments may be designed and performed to testwhether a single dose shortly before or concurrent with NNK would besufficient.

Preparation and Characterization of Three Kava Fractions

Kava from Gaia Herbs was separated into three fractions—Fractions A, B,and C with nineteen repeates. The chemical profile of each fraction wascharacterized by ¹H-NMR to ensure the reproducible integrity of eachmodality (FIG. 6). The mass of each fraction was determined (FIG. 6).Fractions A, B, and C accounted for 36.3%, 51.4%, and 9.9% of the massbalance of kava, respectively. The quantitative mass balance (97.7%)suggests that most, if not all, components were recovered. Reconstitutedkava from Fractions A, B, and C also revealed no difference incomposition from the original kava preparation, based on ¹H-NMR and HPLCanalyses (FIGS. 6 and 7). HPLC analyses also showed that Fraction Ccontained chemicals not detectable in traditional kava (FIG. 7).

The major chemicals in Fraction B and C were isolated, characterized by¹H-NMR and mass spectrometry, and abundance determined (FIG. 8). Allthese chemicals have been previously identified from kava products (DukeJ, Dr. Duke's phytochemical and ethnobotanical databases, in, 2013 pp.http://www.ars-grin.gov/duke/.). Fraction B contains six kavalactonesand one flavanone. Fraction C includes two additional flavanones, twobornyl esters, and two chalcone-based flavokawains A and B. Chemicals inFraction A were not characterized because the chemopreventive efficacydata showed that Fraction A was the least efficacious and that GaiaHerbs kava's chemopreventive potential could be recapitulated byFraction B (Table 4).

Estimating Minimum Effective Dosage and Searching for Active FractionAgainst NNK-Induced Lung Adenoma Formation in A/J Mice—Experiment 4

To determine the minimum dose of kava that could effectively inhibitNNK-induced lung adenoma formation in A/J mice, the same carcinogenprotocol as above to initiate tumorigenesis was used. NNK-treated A/Jmice were given diet supplemented with kava at a dose of 5, 2.5, and1.25 mg/g of diet during Day 1-Day 14. The incidence and number ofadenoma on the lung surface at the end of Day 119 were quantified (Table4). Similar to previous results, A/J mice without NNK treatment had lowadenoma incidence (20%) and multiplicity (0.2±0.4 lung adenoma/mouse)while NNK-treated A/J mice had 100% adenoma incidence and high adenomamultiplicity (16.0±5.2 lung adenoma/mouse). Kava treatments, at alldosages, reduced adenoma incidence by 73-87% and reduced adenomamultiplicity by ˜99%. These data suggested that future experiments wouldbe needed to explore even lower dosages to define the minimum effectivedose of kava to block tumor initiation in this model.

In this study, the efficacy of the three kava fractions at a dose of 2.5mg/g of diet was also evaluated to rank their anti-initiation efficacy(Table 4, Groups 6-8). Fraction A, equivalent to kava at a dose of 6.9mg/g of diet based on its abundance in kava, caused no reduction inadenoma incidence and only weakly reduced adenoma multiplicity by 25%(12.0±5.0 lung adenoma/mouse, p<0.01). Fraction B, equivalent to kava ata dose of 4.9 mg/g of diet, reduced adenoma incidence by 93% and reducedadenoma multiplicity to baseline level (0.1±0.5 lung adenoma/mouse,p<0.01). Fraction C, equivalent to kava at a dose of 25.2 mg/g of diet,did not reduce adenoma incidence but reduced adenoma multiplicity by 70%(3.5±2.5 lung adenoma/mouse, p<0.01). None of these regimens causedsignificant bodyweight or liver weight changes in comparison to mice inGroup 2. The data suggest the Fraction B contained the overwhelmingmajority, if not all, of the active compounds, Fraction C containedminor amount whereas Fraction A contained literally none.

Effect of Kava and its Fractions on DNA Damage Induced by NNK in A/JMouse Lung Tissues

Since the data convincingly established the highly selectiveanti-initiation efficacy of kava and its Fraction B against NNK-inducedlung adenoma formation, reduction of NNK-induced DNA damage as aplausible mechanism of chemoprevention was focused on next. Additionalexperiments to collect lung tissues were designed to characterize thetime-course profiles of four NNK-derived DNA adducts (7-pobG, O²-pobdT,O⁶-pobdG and O⁶-mG) in the lung tissues of the A/J mice upon kavaexposure at a dose of 5 mg/g of diet. As expected, no NNK-derived DNAadducts were detected in the negative control mice (data not shown)while significant amounts of all four DNA adducts were detected in micewith NNK treatment (FIG. 1A). Kava treatment reduced the quantity of allfour DNA adducts (FIG. 1A). When the abundance of each DNA adduct wasnormalized relative to its time-controlled NNK-treatment group (FIG.1B), the extents of reduction in 7-pobG, O²-pobdT, and O²-pobdG weresimilar (30-40%), particularly during the first 24 h after NNK treatmentwhen the contribution of DNA repair and intrinsic instability of theseadducts are less important. For O⁶-mG, however, the reduction was 70-80%(FIG. 1B). Because there were no differences in the relative abundanceof any of these four DNA adducts at different time points after NNKtreatment (FIG. 1B), kava-induced reduction in these DNA adducts is morelikely mediated through the inhibition of their formation instead of theactivation of DNA repair mechanisms.

The effect of Fractions A, B, and C at a dose of 2.5 mg/g of diet onNNK-induced DNA adducts 24 h after NNK treatment were evaluated next. Asshown in FIG. 1C, all kava fractions reduced NNK-induced DNA damage.However, the extents of reduction in 7-pobG, O²-pobdT, and O⁶-pobdG werevery similar (FIG. 1D) and had no correlation with their distinctcapacities in blocking lung adenoma formation. The extents of reductionin O⁶-mG, on the other hand, were quite different. Fraction B greatlyreduced O⁶-mG (72%) while Fraction A and C had no effect on O⁶-mG. Theextent of reduction in O⁶-mG correlated with their capabilities inreducing lung adenoma multiplicity (Table 4). These data suggest thatblocking the formation of O⁶-mG (and possibly other methylation adducts)in the lung DNA by active compounds in kava Fraction B was a likelymechanism for its efficacy against NNK-induced lung tumorigenesis inthis model.

DISCUSSION

The results from this study clearly demonstrated that kava, when givenbefore and during NNK treatment period (initiation stage), was highlyefficacious in preventing lung adenoma formation in A/J mice, with a˜99% reduction in adenoma multiplicity at a dose of as low as 1.25 mg/gof diet. The minimum effective dose remains to be defined. Suchtreatments also reduced lung tumor incidence. A similar degree ofchemopreventive effect was maintained even when the studies wereterminated at later stages, suggesting that kava blocks lungtumorigenesis instead of slowing down the process. Furthermore, the dataconvincingly established the feasibility of using kava in as few as 3once-daily bolus treatments (i.e., before, during and after the NNKinjection) to block the adenoma initiation activity of NNK. While thesedata further substantiated the chemopreventive potential of kava againsttobacco-induced lung cancer, they also demonstrated a new paradigm ofhighly effective initiation-stage specificity of kava against thiscarcinogen. Such drastic efficacy has not been reported previously inliterature except for several synthetic derivatives of phenethylisothiocyanate (PEITC) (Alworth et al., Carcinogenesis 1993; 14:1711-3).

On the other hand, kava showed much lower efficacy when its treatmentstarted after the second NNK administration, suggesting that kava atthis dose and format mainly blocks the initiation of NNK-induced lungtumorigenesis. These results differed from those of previous studies,which demonstrated that kava at a dose of 10 mg/g of diet decreased NNK-and BaP-induced adenoma formation in A/J mice in the post-initiationstage (i.e., kava treatment started one day after the final dose ofcarcinogen treatment) as well as in the initiation stage (Johnson etal., Cancer Prev Res (Phila) 2008; 1:430-8). There are severaldifferences between these studies that may account for this apparentdiscrepancy. First of all, the routes of carcinogen administration andthe dose-intensity were different. NNK in this study was given by i.p.injection (2 weekly injections) whereas previous work involved gavage ofNNK and BaP mixture (8 weekly gavages). These may lead to differentmetabolic processing of the carcinogen(s) and reactive metabolitestoward DNA and thereby different pathogenetic alterations in the targettissues in these models. Secondly, the kava treatment regimen in thisstudy (Group 6), most closely mimicking that used in the previous study,displayed a modest reduction in lung adenoma multiplicity (Table 1),albeit to a lesser extent than those in the previous study. This mightbe explained by the dosage difference in these studies. Finally, kavadid not completely block NNK- and BaP-induced lung adenoma formationwhen it was given during the initiation phase even at a dose of 10 mg/gof diet (Johnson et al., Cancer Prev Res (Phila) 2008; 1:430-8).Compared with the high efficacy against NNK-induced initiation in thecurrent study, it is possible that kava is less effective in blockingBaP-induced lung tumor initiation.

In the search for the active compound(s), a highly reproduciblefractionation protocol was developed, separating kava into threefractions. Fraction A contains the polar chemicals, Fraction B containsthe chemicals with intermediate polarity, and Fraction C contains thenon-polar chemicals not detectable in traditional kava. When evaluatedat a dose of 2.5 mg/g of diet, Fractions A and C, equivalent doses muchhigher than kava at 5 mg/g of diet, only weakly reduced lung adenomamultiplicity with no reduction in tumor incidence. Fraction B, on theother hand, completely blocked NNK-induced lung adenoma formation at adose equivalent to kava at 5 mg/g of diet. These data clearlydemonstrate that Fraction B fully recapitulates kava's lungchemopreventive efficacy and contains the active compounds, whereasFractions A and C contain none or little. Six kavalactones have beenidentified in Fraction B, accounting for 94% of its mass balance.Although there had been no report of their efficacy in any in vivotumorigenesis models, these kavalactones are likely responsible forkava's efficacy in blocking NNK-induced lung tumorigenesis in A/J mice.It is noteworthy that Fraction B is free of flavokawains A and B thatmay contribute to kava's hepatotoxic risk (Zhou P, et al., Faseb J.2010; 24:4722-32). Although flavokawains A and B have revealedanticancer activities in several xenograft models (Zi et al., Cancer Res2005; 65:3479-86; Tang Y et al., Int J Cancer 2010; 127:1758-68; Lin etal., J Nutr Biochem 2012; 23:368-78), results from current studiesindicate that they are not the active compounds against NNK-induced lungtumorigenesis initiation, consistent with the results from the previousstudy (Johnson et al., Am J Chin Med 2011; 39:727-42).

Given the highly selective anti-NNK-initiation action of kava and itsfraction B, their effect on NNK-induced DNA damage in lung tissue wascharacterized as a possible mechanism. NNK, an asymmetrical nitrosamine,can be activated to two types of DNA reactive species via differenthydroxylation pathways (FIG. 2). Methyl hydroxylation generates4-oxo-4-(3-pyridyl)-1-butanediazohydroxide, which leads to a panel ofDNA adducts, including 7-pobG, O²-[4-(3-pyridyl)-4-oxobut-1-yl]cytidine(O²-pobC), O²-pobdT, and O⁶-pobdG (Urban et al., Chem Res Toxicol 2012;25:2167-78). Methylene hydroxylation generates methanediazohydroxide,leading to another set of DNA adducts, including 7-methylguanine (7-mG),O⁶-mG, and O⁴-methylthymidine (O⁴-mT). The abundance of methylation DNAadducts are typically 10-20 fold more than those of the pob DNA adducts,likely due to a combination of preferential methylene hydroxylation ofNNK and higher reactivity of methanediazohydroxide intermediate(Peterson et al., Cancer Res 1991; 51:5495-500). Four of these DNAadducts were analyzed in the lung, 7-pobG, O²-pobdT, O⁶-pobdG, andO⁶-mG, because of their better stability, their representation of bothpathways of NNK activation, and their potential tumorigenicity (Urban etal., Chem Res Toxicol 2012; 25:2167-78; Peterson et al., Cancer Res1991; 51:5557-64; Loechler et al., Proc Natl Acad Sci USA 1984;81:6271-5).

Surprisingly, kava treatment causes different extents of reduction inthese four DNA adducts with high preference on O⁶-mG. To our knowledge,kava is the first candidate that demonstrates such a unique mechanism.Given that the POB adducts are generated via methyl hydroxylation of NNKwhile O⁶-mG is generated via methylene hydroxylation (FIG. 2), kavaFraction B chemicals may preferentially inhibit NNK methylenehydroxylation over methyl hydroxylation. It is also possible thatFaction B chemicals better react with and trap methanediazohydroxideover 4-oxo-4-(3-pyridyl)-1-butanediazohydroxide, leading to the observedpreferential reduction in O⁶-mG. Nevertheless, work from Peterson et al.shows that O⁶-mG has a strong and positive correlation with lung tumormultiplicity in A/J mice (Peterson et al., Cancer Res 1991; 51:5557-64).A/J mice with increased DNA repair capacity specific to O⁶-mG are lesssusceptible to NNK-induced lung tumorigenesis as well (Liu et al.,Carcinogenesis 1999; 20:279-84). In addition, the miscoding propertiesof O⁶-mG have been well established (Loechler et al., Proc Natl Acad SciUSA 1984; 81:6271-5). These results argue for the high tumorigenicity ofO⁶-mG relative to the POB adducts and the possible cause-effect of itsreduction by kava and Fraction B chemicals to their impressiveanti-initiation efficacy.

In summary, kava blocks NNK-induced lung tumorigenesis in A/J mice withhigh selectivity for the initiation stage. Mechanistically kava FractionB chemicals preferentially reduces NNK-induced O⁶-mG DNA adduct in thelung tissues. These results also suggest that kavalactones in Fraction Bmay be the active compounds.

Example 2 Effect of Dietary Kava Fraction B Supplement on Carcinogenesisin Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) MiceIntroduction of the Model

In the TRAMP model, the rat probasin promoter drives transgenic“prostate-specific” expression of simian virus 40 (SV40) T-antigen(T-Ag) and small t-antigen; T-Ag binds and inactivates theretinoblastoma (Rb) and p53 tumor suppressor genes; and small t-antigenalso drives cell proliferation (Greenberg et al, PNAS, 1995).

There are at least 2 distinct lineages of carcinogenesis in this model(Chiaverotti et al, Am J Pathol, 2008; Huss et al, Neoplasia, 2007, Wanget al, Prostate, 2011). One lineage is the androgen receptor(AR)-dependent glandular epithelial lesions, which usually involve thedorsal-lateral prostate (DLP) lobes, less for anterior (AP) and ventral(VP) lobes. The other lineage is the AR-independent neuroendocrine(NE)-like poorly differentiated (PD) carcinomas: life-time NE-Caincidence=⅓ in C57BL/6 mice, usually arise from the ventral prostate(VP) lobes, smallest of all lobes. The weight of genito-urinary (GU)track (bladder, seminal vesicles, prostate lobes) is conventionally usedas a measure of tumor load. Weight of DLP lobes is more specific forepithelial lesion expansion.

Research Design and Safety Monitoring

The detailed experimental design is listed in Table 5. The experimentalprotocols are included below. Specifically, AIN 93M purified powdereddiet was supplemented with 0.4% of KAVA extract fraction B (see, Example1 and the Figures). Fresh diet was prepared once a week, made into moistcookies and air dried and stored at 4 degrees C. Cookies were weighed inand out for estimating food intake by food dis-appearance data. Freshdiet provided twice a week. Food intake=approximately 3 g/mouse daily.Mice health was monitored 2-3 times initially; however, this was donemore frequently when the mice were over 16 wks. Mice body weight wasmeasured once a week initially; then bi-weekly.

Results and Conclusions

As shown in FIGS. 9-12 and Tables 6 and 7, feeding 0.4% Kava extractFraction B from 8 weeks of age (early PIN lesions) in TRAMP micesignificantly decreased genito-urinary tract weight and prostate weightat 16 and 28 weeks of age, respectively. Such dietary treatmentdecreased incidence of NE-carcinomas at 28 weeks from 50% to 14.3% (X²test, p<0.05). Kava Fraction B inhibited both epithelial lesion lineageand NE-carcinogenesis lineage.

Example 3 Effect of Dihydromethysticin and Kava Fraction B on LungCancer Treatment in the Xenograft Models Introduction of the Models

Cancer xenograft models inoculated using human cancer cell lines havebeen well-accepted as the animal model to evaluate and develop cancertherapeutic agents. Human H2009 and A549 lung cancer cell lines are twogeneral cancer cell lines for establishing xenograft models. In thisexample, kava and its modalities, including Fractions A, B, C,dihydromethysticin, and methysticin, have been evaluated for theirpotential to suppress tumor growth, which support the potential use ofthese compounds/compositions as therapeutic agents for the treatment ofhuman cancers (see, Example 1 for a description of Fractions A, B andC).

Research Design and Safety Monitoring

Typically human origin lung cancer cells were cultured and mixed withMetrigel. 2-5×10⁶ cells were inoculated in the nude mice. Mice wererandomized into different groups when the tumor size reached˜50 mm³.Treated mice received drug candidate at a dose of 40 mg/kg bodyweightvia i.p. injection or oral gavage. The tumor size was monitored once ortwice a week until 5 weeks since the treatment or when the size of thetumors >1000 mm³. Mouse bodyweight was monitored once a week. Mice wereCO² euthanized and the tumors were collected and weighed. Major organswere grossly examined and weighed as well.

Results and Conclusion

As shown in FIG. 13, dihydromethysticin treatment significantly reducedthe tumor volume induced by H2009 human lung cancer cell lines relativeto control group, suggesting that dihydromethysticin is a promisinganticancer agent. Similarly as shown in FIGS. 14 and 15, Fraction B,which contains dihydromethysticin also significantly reduced the tumorvolume induced by A549 human cancer cell line relative to controlgroups, suggesting that Fraction and likely dihydromethysticin arelikely anticancer agents against different malignancies.

Example 4 Dihydromethysticin (DHM) from Kava Blocks Tobacco Carcinogen4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Induced LungTumorigenesis and Differentially Reduces DNA Damage in A/J Mice Abstract

As discussed herein, kava and its flavokavain-free Fraction B completelyblocked 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-inducedlung tumorigenesis in A/J mice with a preferential reduction inNNK-induced O⁶-methylguanine (O⁶-mG). In this study, natural(+)-dihydromethysticin (DHM) was identified as a lead compound throughevaluating the in vivo efficacy of five major compounds in Fraction B onreducing O⁶-mG in lung tissues. (+)-DHM demonstrated outstandingchemopreventive activity against NNK-induced lung tumorigenesis in A/Jmice with 97% reduction of adenoma multiplicity at a dose of 0.05 mg/gof diet (50 ppm). Synthetic (±)-DHM was equally and possibly moreeffective as the natural (+)-DHM in these bioassays while a structurallysimilar analog, (+)-dihydrokavain (DHK), was completely inactive,revealing a sharp in vivo structure-activity relationship (SAR).Analyses of an expanded panel of NNK-induced DNA adducts revealed thatDHM reduced a subset of DNA adducts in lung tissues derived from4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the activemetabolite of NNK). Preliminary 17-week safety studies of DHM in A/Jmice at a dose of 0.5 mg/g of diet (at least 10× its minimum effectivedose) revealed no adverse effects, suggesting that DHM is likely free ofkava's hepatotoxic risk. These results demonstrate the outstandingefficacy and promising safety margin of DHM in preventing NNK-inducedlung tumorigenesis in A/J mice, with a unique mechanism of action andhigh target specificity.

Introduction

Lung cancer causes ˜160,000 deaths in the U.S. and 1.4 million deathsworldwide annually. Due to the limited success in treatment (Cohen, V etal. (2004) Curr. Opin. Pulm. Med. 10: 279-283), prevention is ofparamount importance. Tobacco cessation would, without a doubt, be theideal strategy and should be highly promoted since cigarette smokingcauses 90% of lung cancers. Quitting, however, is very challengingbecause of the addictive nature of nicotine in tobacco products; manysmokers will not succeed even after multiple attempts and with the bestcessation support. These individuals contribute significantly to theestimated 200,000 lung cancer incidence in the U.S. each year inassociation with an estimated annual medical cost of $12 billion.Chemopreventive agents hence need to be developed for these high-riskpopulations, besides further optimizing tobacco cessation methods.During the past few decades, a number of compounds have been identifiedas potential lung cancer chemopreventive agents and several of them havebeen evaluated in the clinic, but unfortunately with no success (Hecht,S S et al. (2009) Nat. Rev. Cancer. 9: 476-488). The lack of clinicaleffectiveness is at least partly due to the moderate in vivo efficacy ofsome of the leads. Novel entities with superior efficacy and uniquemechanisms, therefore, need to be developed.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is atobacco-specific and highly potent pulmonary carcinogen, selectivelyinducing lung adenoma and adenocarcinoma in various species (Hecht, S S.(1998) Chem. Res. Toxicol. 11: 559-603). Substantial evidence alsosuggests that NNK in cigarette smoke contributes to pulmonaryadenocarcinoma in the U.S. smokers. The incidence of lung adenocarcinomahas increased in the U.S. during the past few decades. Such a change hasbeen observed only among smokers, suggesting its association withcigarette related factors. One study noted an increase in NNK levels inthe mainstream smoke of a leading U.S. non-filter cigarette between 1978and 1992 while levels of benzo[a]pyrene (BaP), anotherwell-characterized pulmonary carcinogen, decreased in the same cigarettefrom 1959-1992 (Hoffmann, D et al. (1993) Journal of Smoking-relateddisorders. 4: 165-189). Further evidence supporting a role for NNK inhuman lung adenocarcinoma derives from the comparison of NNK content incigarette smoke and adenocarcinoma rates among the U.S., UK, Canada andAustralia (Burns, D M et al. (2011) Cancer Causes Control. 22: 13-22).

Mechanistically NNK is metabolically activated via α-hydroxylation togenerate two reactive species, leading to two types of DNAmodifications—methylation and pyridyloxobutylation (Hecht, S S. (2012)Int. J. Cancer. 131: 2724-2732; Hecht, S S. (2008) Chem. Res. Toxicol.21: 160-171) (FIG. 16). The extent of DNA damage can be characterized byquantifying different DNA adducts—O⁶-methylguanine (O⁶-mG) and 7-methylguanine (7-mG) for methylation and7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-pobG),O⁶-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine (O⁶-pobdG) andO²-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O²-pobdT) forpyridyloxobutylation. NNK is also metabolically converted to4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) (FIG. 16). NNAL canbe activated via α-hydroxylation as well to generate two reactivespecies, resulting in two types of DNA modifications—methylation andpyridylhydroxybutylation. The extent of pyridylhydroxybutylation can becharacterized by quantifying 7-[4-(3-pyridyl)-4-hydroxobut-1-yl]guanine(7-phbG), O⁶-[4-(3-pyridyl)-4-hydroxobut-1-yl]-2′-deoxyguanosine(O⁶-phbdG) and O²-[4-(3-pyridyl)-4-hydroxobut-1-yl]thymidine (O²-phbdT).DNA damage by NNK and NNAL has been well established as one majorunderlying mechanism for NNK-induced lung tumorigenesis (Hecht, S S.(1998) Chem. Res. Toxicol. 11: 559-603). Reducing such DNA damage is,therefore, a plausible strategy for chemoprevention of lung cancer.

The A/J mouse NNK-induced lung tumorigenesis model has been widely usedin evaluating lung cancer chemopreventive agents (Hecht, S S et al.(2009) Nat. Rev. Cancer. 9: 476-488) because A/J mice are prone to lungtumorigenesis (O'Donnell, E P et al. (2006) Cancer Lett. 241: 197-202)and the tumors have similar morphological, histological and molecularfeatures as human lung adenocarcinomas (Malkinson, A M. (2001) LungCancer. 32: 265-279). With NNK treatment, A/J mice develop lung tumorswith a 100% incidence and a high multiplicity (Hecht, S S et al. (2009)Nat. Rev. Cancer. 9: 476-488; Malkinson, A M. (2001) Lung Cancer. 32:265-279). Among NNK-induced DNA adducts, the quantity of O⁶-mG has showna strong positive correlation with lung tumor multiplicity (Peterson, LA et al. (1991) Cancer Res. 51: 5557-5564) and A/J mice with anincreased DNA repair capacity specific to O⁶-mG are less susceptible toNNK-induced lung tumorigenesis (Liu, L et al. (1999) Carcinogenesis. 20:279-284). In combination with the high miscoding property of O⁶-mG(19-22), NNK-induced O⁶-mG is believed essential to initiate lungtumorigenesis in A/J mice.

As described herein, a commercial kava product efficiently blocksNNK-induced lung tumorigenesis in A/J mice at a dose of 1.25 mg/g ofdiet (Leitzman, P et al. (2014) Cancer. Prev. Res., 7: 86-96). FractionB of this product, containing mostly kavalactones (FIG. 17A), fullyrecapitulates its chemopreventive efficacy. Kava and Fraction B reducesNNK-induced DNA damage in the lung with a preference in O⁶-mG adductover the POB adducts while ineffective fractions have no effect on O⁶-mG(Leitzman, P et al. (2014) Cancer. Prev. Res., 7: 86-96). Monitoring theimpact of single chemicals in Fraction B on O⁶-mG, therefore, would bean economic strategy to identify the active lead(s). It is alsomechanistically intriguing regarding the preferential reduction in O⁶-mGadduct.

Thus, described herein is the identification and confirmation ofdihydromethysticin (DHM) as the active compound. DHM potently andefficiently blocks NNK-induced lung tumorigenesis in A/J mice. Asdescribed below, the mechanism leading to its differential reduction inNNK-induced DNA damage has also been investigated and preliminarylong-term safety of DHM in A/J mice upon high-dose exposure is provided.

Abbreviations

DHM, dihydromethysticin; DHK, dihydrokavain; NNK,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNAL,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; BaP, benzo[a]pyrene;O⁶-mG, O⁶-methylguanine; 7-mG, 7-methylguanine; 7-pobG,7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine; O⁶-pobdG,O⁶-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine; O²-pobdT,O²-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine; 7-phbG,7-[4-(3-pyridyl)-4-hydroxobut-1-yl]guanine; O⁶-phbdG,O⁶-[4-(3-pyridyl)-4-hydroxobut-1-yl]-2′-deoxyguanosine; O²-phbdT,O²-[4-(3-pyridyl)-4-hydroxobut-1-yl]thymidine; AGT, O⁶-alkylguanine-DNAalkyltransferase; ANOVA, analysis of variance; ALP, alkalinephosphatase; ALT, alanine aminotransferase; AST, aspartateaminotransferase; BUN, blood urea nitrogen; CK, creatine kinase; H&E,hematoxylin and eosin; MTD, maximum tolerated dose; PEITC, phenethylisothiocyanate; I3C, indole-3-carbinol.

Materials and Methods Chemicals, Reagents and Animal Diets

NNK was prepared by following a reported procedure (Hecht, S S et al.(1983) Carcinogenesis. 4: 305-310). [CD₃]O⁶-mG was purchased fromToronto Research Chemicals (Toronto, Ontario, Canada).[¹³CD₃]7-methylguanine was prepared by following a reported procedure(Peterson, L A et al. (2013) Chem. Res. Toxicol. 26: 1464-1473). 7-PobG,O⁶-pobdG, O²-pobdT, 7-phbG, O⁶-phbdG, O²-phbdT and the corresponding[pyridine-D₄] analogues were synthesized by following reportedprocedures (Lao, Y et al. (2006) Res. Toxicol. 19: 674-682; Sturla, S Jet al. (2005) Chem. Res. Toxicol. 18: 1048-1055; Upadhyaya, P et al.(2008) Chem. Res. Toxicol. 21: 1468-1476). Micrococcal nuclease andphosphodiesterase II were purchased from Worthington BiochemicalCorporation (Lakewood, N.J.). Alkaline phosphatase was purchased fromRoche Molecular Biochemicals (Indianapolis, Ind.).

Kava was acquired from Gaia Herbs, Inc. (Brevard, N.C.) as an ethanolextract of the wild crafted lateral root from Vanuatu. It wasstandardized to 150 mg/mL total kavalactones. The AIN-93 G and Mpowdered diets were purchased from Harlan Teklad (Madison, Wis.). Forthe short-term DNA damage study, the AIN-93 G diet was used during theexperimental period. For the long-term lung tumorigenesis study, theAIN-93 G diet started one week before the first dose of NNK and endedone week after the second dose of NNK. The AIN-93 M diet was used duringthe rest of the experimental period.

Isolation of Chemicals in Fraction B

Five kavalactones (desmethoxyyangonin, (+)-dihydrokavain (DHK),(+)-kavain, (+)-DHM, and (+)-methysticin, FIG. 17A) were isolated fromFraction B by normal phase silica gel chromatography with a gradientmixture of hexane and ethyl acetate as the eluent. Their chemicalstructures were characterized by ¹H-NMR and mass spectrometry. Theirpurities were estimated >90% by HPLC on a Beckman Coulter System Gold126 solvent module with a 168 detector. A Clipeus C-18 column (5 μm,250×4.6 mm) was used for the analyses. The flow rate was 1 mL/min. Themobile phase A was 10 mM NH₄OAc aqueous solution and B was acetonitrile.The time program used for the analyses was 50% B for 15 min.

Synthesis of (±)-DHM

(±)-DHM was synthesized by following a procedure similar as previouslyreported (Shaik, A A, Tan, J., Lu, J., Xing, C. (2012) ARKIVOC. viii:137-145). Its structure and purity were characterized by ¹H-NMR, massspectrometry and HPLC by following the conditions detailed above.

Diet Preparation

Kava was reconstituted in absolute ethanol (50 mL) and then mixed withthe AIN-93 diet (150 g). Similarly, absolute ethanol (50 mL each)reconstituted with kavalactones was mixed with the AIN-93 diet (150 geach) for the kavalactone diets. Absolute ethanol (50 mL) was mixed withthe AIN-93 diet (150 g) for the control diet. The reconstituted dietswere dried under vacuum to remove ethanol and then ground into finepowders. All diets were mixed with additional AIN-93 diet to the desireddose.

General Protocols for Animal Studies

Female A/J mice (5-6 weeks of age) were purchased from the JacksonLaboratory (Bar Harbor, Me.) and handled according to IACUC-approvedanimal welfare protocols at the University of Minnesota. Upon arrival,the mice were housed in the specific pathogen-free animal facilities ofthe Research Animal Resources at the University of Minnesota.

General Protocols for Isolating DNA from Lung and Liver Tissues

DNA was isolated and purified from ˜100 mg lung and liver tissues ofeach mouse, following Genomic-tip 100/G protocol from Qiagen Corp(Valencia, Calif.).

Assessing the Effect of Five Natural Kavalactones in Fraction B onNNK-Induced O⁶-mG and Three POB Adducts in A/J Mouse Lung Tissues

After one-week acclimation, A/J mice were weighed and randomized intoseven groups (3 mice per group) and switched to the AIN-93 G diet on adate defined as Day 1. Mice in Groups 1 and 2 were given the AIN-93 Gdiet during Day 1-Day 7. Mice in Groups 3-7 were given the AIN-93 G dietsupplemented with desmethoxyyangonin, (+)-DHK, (+)-kavain, (+)-DHM, or(+)-methysticin respectively, at a dose of 1 mg/g of diet during Day1-Day 7. Mice in Groups 2-7 were given a single dose of NNK at 100 mg/kgbodyweight in saline (0.1 ml) via intraperitoneal (i.p.) injection atthe beginning of Day 7 while mice in Group 1 were given saline (0.1 ml).Mice were euthanized by CO₂ overdosing 24 h after NNK treatment, andlung tissues were collected and stored at −80° C. until DNA isolation. Anumber of NNK-induced DNA modifications (O⁶-mG, 7-pobG, O⁶-pobdG andO²-pobdT) were quantified by following a reported procedure (Leitzman, Pet al. (2014) Cancer. Prev. Res., 7: 86-96).

Evaluating the Dose-Response Effect of Kava, (+)-DHM Vs. (+)-DHK andSynthetic (±)-DHM on NNK-Induced DNA Adducts in A/J Mouse Lung and LiverTissues

After one-week acclimation, A/J mice were weighed and randomized intoten groups (3 mice per group except for Group 1 that had one mouse asthe negative control) and switched to the AIN-93 G diet on a datedefined as Day 1. Mice in Groups 1 and 2 were given the AIN-93 G dietduring Day 1-Day 7. Mice in Groups 3-10 were given the AIN-93 G dietssupplemented with kava, (+)-DHK, (+)-DHM, and synthetic (±)-DHMrespectively at the specified dose during Day 1-Day 7. Mice in Groups2-10 were given a single dose of NNK at 100 mg/kg bodyweight in saline(0.1 ml) via i.p. injection at the beginning of Day 7 while the mouse inGroup 1 was given saline (0.1 ml). Mice were euthanized by CO₂overdosing 24 h after NNK treatment, and lung and liver tissues werecollected and stored at −80° C. until DNA isolation. A set ofNNK-induced DNA adducts (O⁶-mG, 7-mG, 7-pobG, O⁶-pobdG, O²-pobdT,7-phbG, O⁶-phbdG and O²-phbdT) were quantified by following reportedprocedures (Leitzman, P et al. (2014) Cancer. Prev. Res., 7: 86-96;Peterson, L A et al. (2013) Chem. Res. Toxicol. 26: 1464-1473;Upadhyaya, P et al. (2008) Chem. Res. Toxicol. 21: 1468-1476). For 7-mGisolation, DNA (30 μg) was dissolved in 10 mM sodium phosphate buffer,pH 7 (500 μL) and spiked with [¹³C₁ ²H₃]7-mG (10 pmol). Samples wereheated to 80° C. for 30 minutes. An aliquot (100 μL) was removed for7-mG analysis.

Assessing the Efficacy of (+)-DHM Vs. (+)-DHK and Synthetic (±)-DHMAgainst NNK-Induced Lung Adenoma Formation in A/J Mice

After one-week acclimation, mice were weighed, randomized into eightgroups and switched to the AIN-93 G diet on a date defined as Day 1. Thenumber of mice in each group is specified in the Results Section (Table8). Mice were fed diets supplemented with different compounds at thedose specified in the Results Section during Day 1-Day 14. On Day 7 andDay 14, mice in the negative control group received 0.1 mL of salinesolution while mice in the other groups received NNK (100 and 67 mg/kg,respectively, in 0.1 mL of saline solution) via i.p. injection. At theend of Day 21, mice were switched to the AIN-93 M diet until the end ofthe study. The diet was replenished twice weekly. The diet consumptionwas measured twice weekly and the bodyweight was monitored weekly. Thisstudy was terminated at the end of Day 119. All mice were euthanizedwith CO₂ overdosing. The lungs were collected and tumors on the surfaceof the lungs were counted under blinded conditions by an A.C.V.P boardcertified pathologist (M.G. O'S.).

Assessing A/J Mouse Safety Upon 17-Week Continuous Exposure of (+)-DHMat a Dose of 0.5 Mg/g of Diet

After one-week acclimation, A/J mice were weighed and randomized intotwo groups (five mice in the control group and ten mice in (+)-DHMtreatment group). Mice in the control group were maintained on theAIN-93 G diet for three weeks and then switched to the AIN-93 M dietuntil the end of Week 17. Mice in (+)-DHM treatment group weremaintained on the same diet as the control group with supplementation of(+)-DHM at a dose of 0.5 mg/g of diet. Food was replenished twiceweekly. Food intake was monitored twice a week and mouse bodyweight wasmonitored once a week. At the end of Week 8, serum samples from eachmouse were prepared from blood collected via a facial vein and analyzedfor a panel of clinical chemistry analytes. At the end of Week 17, micewere euthanized by CO₂ overdosing. Blood was collected with one portionanalyzed for hematology and the other portion processed for serum andanalyzed for a full panel of clinical chemistry analytes. Individualanimal body weights and major organ weights (lung, liver, heart, spleenand kidney) were recorded. Lung, liver, heart, spleen, kidney andpancreas were fixed in 10% neutral-buffered formalin solution.Appropriately fixed tissues were processed into paraffin blocks usingstandard histological techniques, and 5μ sections were cut and stainedwith hematoxylin and eosin (H&E). Histological slides were examinedusing light microscopy by an experienced A.C.V.P board certifiedpathologist (M.G. O'S.).

Statistical Analyses

Data on lung adenoma multiplicity were reported as mean±SD (n=5−9).One-way analysis of variance (ANOVA) was used to compare means among theNNK and NNK/treatment groups. Dunnett's test was used for comparisons ofthe number of tumors between the NNK control and treatment groups whenthe one-way ANOVA analysis was statistically significant. p-value ≦0.05was considered statistically significant. For DNA damage studies,one-way ANOVA was used to compare means±SD (n=3). Dunnett's test wasused for comparisons between the NNK control and treatment groups whenone-way ANOVA analysis was statistically significant. p-value ≦0.05 wasconsidered statistically significant. For the safety study, an unpairedStudent t-test was used for comparison between the control and (+)-DHMtreatment groups. A two-tailed p-value ≦0.05 was consideredstatistically significant. All analyses were conducted using GraphPadPrism 4 from GraphPad Software, Inc. (La Jolla, Calif.).

Results

The Effects of Five Kavalactones in Fraction B on NNK-Induced O⁶-mG,7-pobG, O⁶-pobdG and O²-pobdT in A/J Mouse Lung Tissues

As previous data described herein showed that the reduction of O⁶-mGreduction in lung tissues by different kava entities correlatedpositively with their lung tumor prevention efficacy and Fraction Brecapitulated kava's chemopreventive effect on NNK-induced lungtumorigenesis in A/J mice (Leitzman, P et al. (2014) Cancer. Prev. Res.,7: 86-96), this experiment was designed to identify the key compound(s)in Fraction B that reduced NNK-induced O⁶-mG, which may be the activechemopreventive agent.

Five known kavalactones have been isolated from Fraction B as its majorcomponents (FIG. 17A), accounting for 93% of its mass balance (Leitzman,P et al. (2014) Cancer. Prev. Res., 7: 86-96). These five compounds wereinvestigated for their effect on NNK-induced DNA damage in lung tissues,including O⁶-mG and three POB adducts (7-pobG, O⁶-pobdG and O²-pobdT)(FIG. 17B). Two compounds, (+)-DHM and (+)-methysticin, significantlyreduced NNK-induced O⁶-mG while the other compounds did not, suggesting(+)-DHM and (+)-methysticin as the potential active compounds and theimportance of the 1,3-dioxol functional group. All five compounds hadmuch weaker reduction in the POB adducts (FIG. 17B), consistent withthat of Fraction B (Leitzman, P et al. (2014) Cancer. Prev. Res., 7:86-96). Although not statistically significant, the extent of O⁶-mGreduction by (+)-DHM was greater than that by (+)-methysticin. (+)-DHMwas therefore selected as the lead for further investigation with DHK asa negative control because of its higher structural similarity to(+)-DHM relative to desmethoxyyangonin and (+)-kavain.

The Dose-Response Effect of (+)-DHM on an Expanded Panel of NNK-InducedDNA Damage in Comparison to Kava, (+)-DHK and Synthetic (±)-DHM in A/JMouse Lung Tissues

This study was designed to address three questions. First, what would bethe dose of (+)-DHM to induce a similar extent of O⁶-mG reduction askava did at a dose of 1.25 mg/g of diet, which effectively blockedNNK-induced lung tumor formation (Leitzman, P et al. (2014) Cancer.Prev. Res., 7: 86-96)? Kava was evaluated at two dosages—5 and 1.25 mg/gof diet respectively while (+)-DHM was evaluated at four dosages—1,0.25, 0.1 and 0.01 mg/g of diet with (+)-DHK evaluated at a dose of 1mg/g of diet as a control. Secondly, given that the isolated (+)-DHMcontained impurities, how can the impurity be ruled out as the activecompound? Synthetic (±)-DHM therefore was prepared and evaluated at adose of 1 mg/g of diet. Lastly, what is the mechanism leading to(+)-DHM's preferential reduction in O⁶-mG over POB adducts in lungtissues? Based on the molecular bases of NNK metabolism and DNA damage(FIG. 16) (Hecht, S S. (1998) Chem. Res. Toxicol. 11: 559-603), fourpossible mechanisms were identified: 1) preferential inhibition of NNKmethylene hydroxylation over its methyl hydroxylation; 2) an increasedO⁶-alkylguanine-DNA alkyltransferase (AGT)-mediated O⁶-mG repair(Belinsky, S A et al. (1988) Carcinogenesis. 9: 2053-2058; Peterson, L Aet al. (1993) Cancer Res. 53: 2780-2785); 3) preferential inhibition ofNNAL hydroxylation over NNK hydroxylation; or 4) increaseddetoxification of NNAL. These mechanisms could be differentiated bycharacterizing the effect of DHM on an expanded panel of DNA adducts,including POB DNA adducts (7-pobG, O⁶-pobdG and O²-pobdT), PHB DNAadducts (7-phbG, O⁶-phbdG and O²-phbdT) and methyl DNA adducts (O⁶-mGand 7-mG).

Consistent with previous results, kava preferentially anddose-dependently reduced O⁶-mG in lung tissues (FIG. 18A.a) while it hadminimal effects on POB adducts (FIG. 18A.b-d). (+)-DHM alsopreferentially and dose-dependently reduced O⁶-mG (FIG. 18A.a) withweaker effects on POB adducts (FIG. 18A.b-d) whereas (+)-DHK did notreduce any of these adducts even at a dose of 1 mg/g of diet. The extentof reduction in O⁶-mG by (+)-DHM at a dose of 0.1 mg/g of diet wascomparable to that induced by kava at a dose of 1.25 mg/g of diet (FIG.18A.a). Since (+)-DHM at a dose of 0.01 mg/g of diet had no effect onO⁶-mG, the minimum chemopreventive dose of DHM would be between 0.01-0.1mg/g of diet. (+)-DHM also dose-dependently reduced 7-mG (FIG. 18B.a)with the extents of reduction similar to those in O⁶-mG (FIG. 18A.a).These results indicate that AGT-mediated O⁶-mG repair is not involved inDHM's DNA damage reduction. Surprisingly, (+)-DHM dose-dependentlyreduced PHB adducts (FIG. 18B.b-d) in lung tissues as well, again withthe extents of reduction similar to those in O⁶-mG and 7-mG (FIGS. 18A.aand 18B.a). Since (+)-DHM has minimal effect on POB adducts, theseresults suggest that its reduction in NNK-induced DNA damage is likelymediated through the NNAL pathway, either by inhibiting its activationor enhancing its detoxification (A and B in FIG. 16). The synthetic(±)-DHM at 1 mg/g of diet had similar effects in reducing DNA damage asthe natural (+)-DHM (FIGS. 18 A and B), confirm DHM as the activecompound.

The Lack of Effect of Kava, (+)-DHM, (+)-DHK and Synthetic (±)-DHM onNNK-Induced DNA Damage in A/J Mouse Liver Tissues

Since liver is the major metabolizing organ of the body, we alsocharacterized the effect of kava, (+)-DHM, (+)-DHK and (±)-DHM onNNK-induced DNA damage in liver tissues. Surprisingly none of thetreatments had significant effects on NNK-induced DNA damage in theliver tissues (data not shown).

The Efficacy of Kava, (+)-DHM, (+)-DHK and Synthetic (±)-DHM onNNK-Induced Lung Adenoma Formation in A/J Mice

To validate the chemopreventive potential of DHM, we carried out anNNK-induced lung adenoma assay as shown in Table 8. A/J mice without NNKtreatment had low adenoma incidence (20%) and low adenoma multiplicity(0.2±0.4 lung adenoma/mouse, Group 1) while NNK-treated A/J mice had100% adenoma incidence and high adenoma multiplicity (13.9±6.9 lungadenoma/mouse, Group 2). Kava at a dose of 1.25 mg/g of diet slightlyreduced adenoma incidence by 20% but significantly reduced adenomamultiplicity by 95.6% (to 0.8±0.4, Group 3). Based on the estimatedminimum effective dose of (+)-DHM between 0.01 and 0.1 mg/g of diet, wetested it at 0.5 and 0.05 mg/g of diet (Groups 4 and 5). (+)-DHM at bothdosages reduced adenoma incidence by 40% and significantly reducedadenoma multiplicity by 97.1% (to 0.6±0.5, Groups 4 and 5), suggestingthat its minimum effective dose has not been reached. All mice wereadenoma free upon (±)-DHM treatment at a dose of 0.5 mg/g of diet (Group8). (+)-DHK, as a negative control, showed no reduction at all at 0.5 or0.05 mg/g of diet (Groups 6 and 7). Overall, these data established DHMas a lung cancer chemopreventive agent with a minimum effective doselower than 0.05 mg/g (50 ppm).

A/J Mouse Safety Assessment Upon 17-Week Continuous Exposure of (+)-DHMat a Dose of 0.5 Mg/g of Diet

Cognizant of the hepatotoxic concerns associated with kava (Rowe, A etal. (2012) Res. 26: 1768-1770; Teschke, R et al. (2013) Phytother Res.27: 472-474), we characterized a number of parameters to assess thesafety of (+)-DHM. Mice with (+)-DHM feeding started with a slightlyhigher average bodyweight and remained heavier than the control micethrough the study, but none of the differences were statisticallysignificant (FIG. 20). The food intake was similar as well (FIG. 20).The clinical chemistry results for the serum samples collected at theend of Week 8 are summarized in FIG. 21A, and they included alkalinephosphatase (ALP), alanine aminotransferase (ALT), aspartateaminotransferase (AST), blood urea nitrogen (BUN), albumin, totalbilirubin, creatine kinase (CK), glucose, cholesterol and amylase. Allof these parameters reflect liver functions, except for amylase (amarker for pancreas). No significant differences were detected in any ofthese parameters between the control group and the (+)-DHM group. Theclinical chemistry results of the serum samples collected at the end ofWeek 17 are summarized in FIG. 21B. Same as the 8-week outcome, therewere no significant differences for any of these parameters between thecontrol group and the (+)-DHM group. The time-course changes of theseparameters for each mouse also revealed no obvious difference betweenthe control and (+)-DHM groups (data not shown). On Week 17, theconcentration of various salts in the serum was quantified as well;these data were used to calculate serum osmolality and anion gap, whichrevealed no differences between the control and (+)-DHM groups either(data not shown). The blood samples on Week 17 were also analyzed forfull-panel hematology (one blood sample from a control mouse clotted andcould not be analyzed, which resulted in four data points for thecontrol group, FIG. 22). None of these parameters were significantlydifferent between the control and (+)-DHM groups. Lastly, the finalbodyweight and the relative weights of five organs (FIG. 19) were notsignificantly different between the control and (+)-DHM groups exceptfor the lung tissues. The average relative lung weight for the controlgroup was significantly higher than that for the (+)-DHM group (p=0.05).An inspection of the data from each mouse revealed that one mouse in thecontrol group had an increased lung weight that was attributed tomultifocal hemorrhage within the lung at the time of euthanasia.Excluding this animal, there were no significant differences between thecontrol and (+)-DHM groups (p=0.10). Histopathological examination ofliver, lung, heart, kidney, spleen and pancreas tissues revealed nosignificant differences between the control and (+)-DHM groups (data notshown).

Discussion and Conclusion

The results described herein have unambiguously identified DHM as apotent chemopreventive agent that safely and completely blocksNNK-induced lung tumorigenesis in A/J mice at a dietary intake dose of0.05 mg/g (50 ppm). (+)-DHM was identified as the lead among fivestructurally similar compounds based on their efficacy to reduce O⁶-mGin lung tissues in a short-term assay (FIG. 17B). In a follow-updose-response experiment using DNA adduct reduction as the readouts, itsminimum effective dose was estimated between 0.01-0.1 mg/g of diet (FIG.18). Therefore (+)-DHM was tested at doses of 0.5 mg/g of diet (500 ppm)and 0.05 mg/g (50 ppm) with a 2-week exposure window during the NNKinitiation period only (Table 8, Groups 4 and 5). Since (+)-DHM at 0.05mg/g reduced NNK-induced lung adenoma multiplicity by 97% and was aseffective as (+)-DHM at 0.5 mg/g, its minimum effective dose wouldlikely be lower than 0.05 mg/g. An extrapolation of an effective dose of0.05 mg/g of diet for a mouse of 20 g (daily food intake 3 g) would beequivalent to a human dose of 47 mg/day (based on 75 kg humanbodyweight) according to the Body Surface Area Normalization method(Reagan-Shaw, S et al. (2008) FASEB J 22: 659-661). Thus from the doseaspect, (+)-DHM is highly feasible for human usage. The results of thesynthetic (±)-DHM are also informative. First, they confirmed DHM as theactive chemopreventive agent. Second, it appeared that the syntheticracemic forms outperformed the natural (+)-DHM in blocking NNK-inducedDNA adducts and lung adenoma formation at the tested dose of 1 mg/g and0.5 mg/g, respectively, although the differences were not statisticallysignificant. It remains to be determined whether the synthetic (−)-DHMmay be more efficacious than the natural (+)-DHM in a dose range closeto its minimum effective dose.

It is interesting to note that (+)-DHK, a structural analog of (+)-DHM(FIG. 17A), was completely inactive in blocking DNA damage (FIGS. 17 and18) or lung adenoma formation (Table 8). Such a sharp in vivostructure-activity relationship (SAR) between (+)-DHM and (+)-DHKsuggests a high target specificity of (+)-DHM. It also highlights theimportance of the 1,3-dioxol functional group in (+)-DHM for itsefficacy. Such a five-member ring may be critical for (+)-DHM tospecifically interact with its molecular target, which remains to beidentified, leading to its outstanding chemopreventive efficacy.Alternatively (+)-DHM may be metabolized via the 1,3-dioxol functionalgroup to generate the in vivo active form. Further investigation isneeded to differentiate these possibilities.

Mechanistically (+)-DHM dose-dependently reduced O⁶-mG, 7-mG, 7-phbG,O⁶-phbdG and O²-phbdT in the lung tissues with similar extents ofreduction (FIGS. 18A.a and 18B.a-d) while it had much weaker effect on7-pobG, O⁶-pobdG and O²-pobdT (FIG. 18A.b-d). These data suggest thatDHM may preferentially block NNAL-mediated DNA damage, possibly viainhibiting NNAL activation (A in FIG. 16) or increasing NNALdetoxification (B in FIG. 16). A closer inspection of the extent ofreduction in methyl and PHB adducts reveals a slightly higher reductionin methyl adducts, although the difference was not statisticallysignificant (FIG. 18C showed a representative set of data with (+)-DHMtreatment at a dose of 1 mg/g of diet). Since the extent of reduction inPOB and PHB adducts are all less than those of the methyl adducts, DHMmay preferentially inhibit methylene hydroxylation over methylhydroxylation of NNK and NNAL as well. The lack of effect of DHM onNNK-induced DNA damage in the liver is intriguing, which may be mediatedvia a potential DHM abundance difference in lung and liver tissues.Alternatively, lung tissues may contain DHM-interacting biomoleculesinvolved in NNAL activation/detoxification that are absent in livertissues.

There have been limited reports characterizing multiple DNA adducts uponNNK treatment in A/J mice in both lung and liver tissues. An early studyby Morse et al. showed that phenethyl isothiocyanate (PETIC) reducedO⁶-mG in lung tissues, but other DNA adducts and liver tissues were notanalyzed (Morse, M A et al. (1989) Cancer Res. 49: 2894-2897). A laterstudy by Prokopczyk et al. showed that synthetic1,4-phenylenebis(methylene)selenocyanate, an analog of PEITC,preferentially reduced O⁶-mG in comparison to 7-mG (Prokopczyk, B et al.(1996) Carcinogenesis. 17: 749-753). Crampsie et al. recently reportedthat phenylbutyl isoselenocyanate (ISC-4), again a synthetic seleniummimic of PEITC, reduced both methyl and POB DNA damage in A/J mouse lungand liver tissues (Crampsie, M A et al. (2011) Cancer Prev. Res. 4:1884-1894). Another promising lung cancer chemopreventive agent,indole-3-carbinol (13C) was found to reduce O⁶-mG in lung tissues butincreased O⁶-mG in liver tissues, potentially via increasing liver NNKmetabolism (Morse, M A et al. (1990) Cancer Res. 50: 2613-2617). Thestudy described herein is the first that characterized all three typesof DNA damage in A/J mice and DHM appears to have a different mechanismrelative to PEITC- and I3C-based lung cancer chemopreventive agents.

More characterizations have been performed on NNK-induced DNA damage inF344 rat lung and liver tissues and several studies have analyzed theimpact of the chirality of NNAL as well (Lao, Y et al. (2006) Chem. Res.Toxicol. 19: 674-682; Upadhyaya, P et al. (2008) Chem. Res. Toxicol. 21:1468-1476; Lao, Y et al. (2007) Chem. Res. Toxicol. 20: 235-245;Upadhyaya, P et al. (2009) Drug Metab. Dispos. 37: 1147-1151). Upadhyayaet al. demonstrated that (R)-NNAL preferentially generated PHB adducts,while (S)-NNAL and NNK produced mainly POB adducts. Although there hasbeen no characterization of (R)- and (5)-NNAL-induced DNA damage in A/Jmice, these two isomers appeared to have different metabolism andtumorigenicity (Upadhyaya, P et al. (1999) Carcinogenesis. 20:1577-1582). It remains to be determined whether the PHB DNA damage inA/J mouse lung tissues mainly derives from (R)-NNAL and whether DHMselectively inhibits the activation of (R)-NNAL, leading to thepreferential reduction in PHB DNA adducts in lung tissues.Alternatively, DHM may selectively enhance the detoxification of(R)-NNAL.

Because of the purported hepatotoxic risk of kava, the safety of (+)-DHMis of great importance. A/J mice continuously exposed to (+)-DHM at adose of 0.5 mg/g of diet (at least ten times its minimum effective dose)for 17 weeks did not present with any adverse effects in the followingparameters: average weekly bodyweight increase, average weekly foodintake, clinical chemistry analyses of serum samples collected at Week 8and Week 17, hematology analysis at Week 17, final bodyweight, relativeweight of heart, lung, liver, kidney and spleen at Week 17, and thepathology of heart, lung, liver, kidney, spleen and pancreas at Week 17.Since the 0.5 mg/g dose appeared to be still below the maximum tolerateddose (MTD, which remains to be determined), (+)-DHM is expected to havea very wide safety margin as a lung cancer chemopreventive agent. Onenon-kavalactone compound in kava has been identified that recapitulatesits hepatotoxic risk while (+)-DHM under the same conditions revealed nosign of hepatotoxic risk, suggesting that (+)-DHM is likely free of thehepatotoxic concern associated with kava (see, Example 5).

In summary, the data described herein support DHM as a potent andefficacious chemopreventive agent against NNK-induced lung tumorigenesisin A/J mice with a unique mechanism of action relative to severalwell-known lung cancer chemopreventive agents. Its sharp in vivo SARsuggests high target specificity, which is highly desirable to minimizeadverse effects as reflected by its impressive safety profile. DHM istherefore a promising lung cancer chemopreventive agent.

Example 5 Flavokawains A and B in Kava, not Dihydromethysticin,Potentiate Acetaminophen-Induced Hepatotoxicity in C57BL/6 Mice Abstract

Anxiolytic kava products have been associated with rare but severehepatotoxicity in humans. This adverse potential has never been capturedin animal models and the responsible compound(s) remains to bedetermined. The lack of such knowledge greatly hinders the preparationof a safer kava product and limits its beneficial applications. In thisstudy, the toxicity of kava as a single entity or in combination withacetaminophen (APAP) in C57BL/6 mice was evaluated. Kava alone revealedno adverse effects for long-term usage even at a dose of 500 mg/kgbodyweight. On the contrary a three-day kava pre-treatment potentiatedAPAP-induced hepatotoxicity, resulted in an increase in serum ALT andAST and increased severity of liver lesions. Chalcone-based flavokawainsA (FKA) and B (FKB) in kava recapitulated its hepatotoxic synergism withAPAP while dihydromethysticin (DHM, a representative kavalactone and apotential lung cancer chemopreventive agent) had no such effect. Theseresults, for the first time, demonstrate the hepatotoxic risk of kavaand its chalcone-based FKA and FKB in vivo and suggest that herb-druginteraction may account for the rare hepatotoxicity associated withanxiolytic kava usage in humans. Kava preparations, such as Fraction Bdescribed herein, which are free of flavokawains A and B, would haveminimized risk and enriched beneficial components.

Introduction

Traditional kava is an aqueous extract of the roots of Piper methysticumand serves as a ceremonious and daily beverage or an herbal remedy forSouth Pacific islanders (Gounder, R. (2006) Pac. Health Dialog 13,131-135). Kava had also been used clinically to treat mild and moderateanxiety based on results of numerous clinical trials (LaPorte, et al.(2011) Hum. Psychopharmacol. 26, 102-111; Sarris, et al. (2009) Hum.Psychopharmacol. 24, 41-48; Sarris, et al. (2009) Psychopharmacology(Berl) 205, 399-407; Pittler, M. H., and Ernst, E. (2000) J. Clin.Psychopharmacol. 20, 84-89). Anxiolytic kava was typically prepared asan organic extract of kava root with ethanol or acetone, instead of thetraditional aqueous preparation. Anxiolytic kava had been banned inEurope and a few other countries since 2002 because of its risk toinduce hepatotoxicity and it is listed on the USA FDA advisory board(Teschke, R., and Wolff, A. (2009) Dig. Liver Dis. 41, 891-901; Teschke,et al. (2013) Phytother. Res. 27, 472-474), but Germany's FederalAdministrative Court negated the ban in June 2014 (Carreno, et al.(2014) FRATINIVERGANO—European Lawyers 13, 2-5).

Various causes have been proposed for kava's hepatotoxic risk but nonehave been validated so far. First of all, in response to high demand,anxiolytic kava may have included non-root toxic plant parts (Schulze,et al. (2003) Phytomedicine 10, 68-73). It has also been postulated thatsome kava roots were not properly dried, resulted in hepatotoxincontamination (Anke, J., and Ramzan, I. (2004) Planta Med. 70, 193-196).Usage of non-traditional cultivars could be another cause; differentkava cultivars have diverse chemical profiles while traditional kava isprepared from only a few of them (Anke, J., and Ramzan, I. (2004) PlantaMed. 70, 193-196; Lebot, et al. (2014) Food Chem. 151, 554-560). Due topreparation difference, traditional and anxiolytic kavas have distinctcomposition profiles (Shaik, et al. (2009) Bioorg. Med Chem. Lett. 19,5732-5736; Leitzman, et al. (2014) Cancer Prev. Res. (Phila) 7, 86-96),which may impose different hepatotoxic risks as well. Furthermore˜90% ofthe purported hepatotoxic cases associated with kava usage involvedconcomitant consumption of other drugs or dietary supplements (W. H.Organization (2007) Assessments of the risk of hepatotoxicity with kavaproducts. WHO Document Production Service; Teschke, R., et al. (2008)Eur. J. Gastroenterol. Hepatol. 20, 1182-1193), suggesting that kava'shepatotoxic risk may be mediated via herb-herb or herb-druginteractions. In addition to kava's anxiolytic benefit, oneepidemiological survey suggested that traditional kava usage may be ableto reduce cancer risk (Steiner, G. G. (2000) Hawaii Med J. 59, 420-422),which was supported by results from several laboratory animaltumorigenesis models (Leitzman, P., et al. (2014) Cancer Prev. Res.(Phila) 7, 86-96; Johnson, et al. (2011) Am. J. Chin. Med. 39, 727-742;Johnson, et al. (2008) Cancer Prev. Res. (Phila) 1, 430-438; Triolet, etal. (2012) Nutr. Cancer 64, 838-846; Zi, X., and Simoneau, A. R. (2005)Cancer Res. 65, 3479-3486; Narayanapillai, et al. (2014) Carcinogenesis35(10), 2365-72). Moreover despite its ban and being on USA FDA'sadvisory list, kava consumption has experienced a global resurgencebased on the amount of kava exported from the major kava producingnations (The Republic of Vanuatu, Fiji, and Tonga) between 2008 and 2013(Martin, et al. (2014) Measuring the chemical and cytotoxic variabilityof commercially available kava. Plos One, Accepted). With the recentoverturn of the kava ban in Germany, its usage is expected to increasefurther globally. Our recent metabolomics and cellular cytotoxicityanalyses of an array of current commercial kava products revealed thatthey were diverse in chemical profile and cellular cytotoxicity (Martin,et al. (2014) Plos One, 9(11): e111572.doi:10.1371/journal.pone.0111572), and likely distinct in their healthbenefit and risk.

Considering the increasing human exposure and the diverse chemicalcomposition of current kava products, the hepatotoxic risk of kava needsto be clarified and the responsible chemicals need to be identified,which is the focus of this study. The results showed that kava was safewhen given alone but significantly enhanced acetaminophen (APAP)-inducedhepatotoxicity in C57BL/6 mice. Chalcone-based flavokawains A (FKA) andB (FKB) recapitulated kava's potentiation of APAP-induced hepatotoxicitywhile dihydromethysticin (DHM) lacked such a risk.

Abbreviations

acetaminophen, APAP; flavokawain A, FKA; flavokawain B, FKB;dihydromethysticin, DHM; analysis of variance, ANOVA; alanineaminotransferase, ALT; aspartate aminotransferase, AST; polyethyleneglycol-400, PEG-400.

Materials and Methods Chemicals and Reagents

An ethanolic extract of the wild crafted kava root from Vanuatu waspurchased from Gaia Herbs, Inc (Brevard, N.C., standardized to 150 mg/mLtotal kavalactones). DHM was purified from this kava product usingnormal phase silica gel chromatography as described earlier(Narayanapillai, et al. (2014) Carcinogenesis 35(10), 2365-72). FKA andFKB were synthesized and characterized following an establishedprocedure (Johnson, et al. (2011) Am. J. Chin. Med. 39, 727-742). Kavaand all compounds were completely dried under vacuum to remove anysolvent residue. APAP was purchased from Sigma Aldrich (MO, St Louis).The desired drug formulations were prepared by mixing kava or purecompounds with PEG-400 and stored at 4° C. until use.

Animal Study Design

All animal studies were performed in compliance with InstitutionalAnimal Care and Use Committee at the University of Minnesota approvalsand guidelines. Six week-old female C57BL/6J mice (Jackson Laboratories,ME) were housed at specific pathogen-free animal facilities of ResearchAnimal Resources, University of Minnesota with free access to standardrodent food and water. All mice were acclimatized for one week beforebeing used for experiments. Mice were gavaged with dose formulations atthe indicated doses and times, euthanized by CO₂ overdosing withnecropsy performed by experienced researchers.

The long-term study was designed to evaluate the hepatotoxicity of kavaalone. C57BL/6 mice were randomized (n=4). Mice in the control groupwere given PEG-400 (200 μL) on a daily basis via gavage, six days aweek, for 14 weeks. Mice in the kava treatment group were given kava ata dose of 500 mg/kg bodyweight on a daily basis via gavage, six days aweek, for 14 weeks. The chosen kava dose was based on the recent safetystudies of another kava product performed by the National ToxicologyProgram (National Toxicology, P. (2012) Natl. Toxicol. Program Tech.Rep. Ser. 1-186). Mouse bodyweight was measured once a week. Uponnecropsy, final bodyweight was measured and serum from each mouse wasanalyzed for alanine aminotransferase (ALT) and aspartateaminotransferase (AST), two major biomarkers of liver function.

The short-term combination studies were designed to evaluate thepotential synergism of kava and its chemicals to APAP inducedhepatotoxicity. C57BL/6 mice were randomized (8-15 mice per group) andwere administered with PEG-400 (200 μL), kava (500 mg/kg bodyweight),DHM or FKA and FKB in PEG-400 (200 μL) at the indicated doses daily viaoral gavage for two days. On the third day, mice in the respectivegroups were co-administered with APAP (800 mg/kg bodyweight) in PEG-400(200 μL). Bodyweight was recorded daily. Necropsies were performed 24hours after the last gavage by experienced researchers. Serum from eachmouse was analyzed for ALT and AST. Livers were collected and preservedin 10% neutral buffered formalin. Appropriately fixed tissues wereprocessed into paraffin blocks using standard histological techniques,and 5 μm sections were cut and stained with hematoxylin and eosin (H&E).Histological slides were examined using light microscopy by anexperienced A.C.V.P board certified pathologist (M.G. O'S.) underblinded conditions, with liver lesions graded on a 0 to 4 scale based onthe extent of necrosis (0=absent, 1=minimal, 2=mild, 3=moderate,4=severe).

Statistical Analysis

The clinical chemistry data were reported as mean±SD (n=4−15). For thelong-term kava alone study, the two-tailed Student t-test was used tocompare the means between the control and treatment groups. p-value≦0.05 was considered statistically significant. One-way analysis ofvariance (ANOVA) was used to compare the means among different groups inthe short-term combination studies. Dunnett's test was used forcomparisons of APAP and other treatment groups when the one-way ANOVAanalysis was statistically significant. p-value ≦0.05 was consideredstatistically significant. All analyses were conducted in GraphPad Prism4 (GraphPad Software, Inc. La Jolla, Calif.).

Results Kava Alone Did not Affect Mouse Growth and Induced No Signs ofHepatotoxicity.

At the tested dose (500 mg/kg bodyweight), daily kava treatment did notaffect mouse growth (data not shown). There were also no statisticallyor biologically significant differences between control and kava-treatedmice with respect to ALT and AST (FIGS. 23A and 23B).

Kava Enhanced APAP-Induced Hepatotoxicity in C57BL/6 Mice.

Since ˜90% of the human kava hepatotoxic cases involved concurrentconsumption of other medications or dietary supplements (W. H.Organization (2007) Assessments of the risk of hepatotoxicity with kavaproducts. WHO Document Production Service; Teschke, R., et al. (2008)Eur. J. Gastroenterol. Hepatol. 20, 1182-1193), herb-drug interactionsmay contribute to kava's hepatotoxic risk. Based on this and on a recentreport that kava enhanced the toxicity of APAP in vitro (Yang, X., andSalminen, W. F. (2011) Phytomedicine 18, 592-600), this study wasdesigned to evaluate the effect of kava on APAP-induced hepatotoxicityin vivo. The treatment regimen was designed to mimic potential scenariosin humans—kava was consumed on a daily basis while APAP was usedoccasionally. As expected kava treatment alone had no effect on ALT andAST while APAP treatment significantly increased serum ALT and ASTactivities (FIG. 24A). Kava and APAP combination caused further increasein serum ALT and AST activities (˜3 fold increase relative to APAPalone, FIG. 24A), and these increases were statistically significant incomparison to APAP treatment alone. Histopathological analyses of theliver tissues revealed no lesions in control and kava treated mice (FIG.24B), confirming the lack of hepatotoxicity by kava treatment alone. Thelesions from APAP-treated mice evenly distributed among differentseverity categories (0 being no lesion and 4 being the highest gradelesion) while kava and APAP combination markedly increased the number ofmice with the highest liver lesion (FIG. 24B), supporting the notionthat the increases in ALT and AST activities were biologicallysignificant. These clinical chemistry data and histopathologicalfindings for the first time demonstrate that kava enhanced APAP-inducedhepatotoxicity in vivo, and may reflect the purported kavahepatotoxicity cases in humans. The histopathological lesion severityalso nicely correlated positively with the clinical chemistry results(FIG. 24C). Therefore only clinical chemistry was performed insubsequent studies.

DHM Did not Potentiate APAP-Induced ALT and AST while FKB IncreasedBoth.

This experiment was designed to explore the potential of DHM and FKB(FIG. 25) to synergize the hepatotoxicity of APAP following the samekava and APAP co-treatment regimen. Thirteen chemicals have beenisolated and quantified from the kava product used in this study with nodetection of pipermethystine (Leitzman, P., et al. (2014) Cancer Prev.Res. (Phila) 7, 86-96). DHM and FKB were selected for this initialevaluation because they are representatives of kavalactones andchalcones respectively, two major classes of chemicals in kava. Inaddition DHM has been recently demonstrated to potently and effectivelyblock NNK-induced lung tumorigenesis in mice (Leitzman, P., et al.Cancer Prev. Res. (Phila) 7, 86-96) while FKB has been identified as themost cytotoxic compound in kava to various cancerous cells (Shaik, etal. (2009) Bioorg. Med. Chem. Lett. 19, 5732-5736; Jhoo, et al. (2006)J. Agric. Food Chem. 54, 3157-3162). The dosages for DHM (37.5 mg/kg)and FKB (11.5 mg/kg) were based on their abundance (7.5% and 2.3%respectively) in this kava product at a dose of 500 mg/kg (Leitzman, etal. (2014) Cancer Prev. Res. (Phila) 7, 86-96). DHM and FKB individuallycaused no effect on serum ALT and AST (FIGS. 26A and 26B). DHM had noeffect on serum ALT and AST as well when combined with APAP (FIGS. 26Aand 26B). FKB on the other hand when combined with APAP moderatelyincreased the serum levels of ALT and AST, and the increase in AST wasstatistically significant (FIGS. 26A and 26B), suggesting that FKBcontributes to kava's potentiation of APAP-induced hepatotoxicity.

The Combination of Flavokawain a (FKA) and FKB Dose-Dependently EnhancedAPAP-Induced Hepatotoxicity.

Given that the kava product used in this study contains flavokawain A(FKA) of similar abundance as FKB (FIG. 25), this experiment wasdesigned to evaluate the dose-response effect of FKA and FKB together onAPAP's hepatotoxicity following the same treatment regimen. The finaldosages of FKA and FKB were 1, 2 and 4 times their abundance (1.6% and2.3% respectively) of a kava dose at 500 mg/kg bodyweight. FKA and FKBtogether did not induce any changes on serum ALT and AST at the threetested dosages (FIGS. 27A and 27B). When combined with APAP, FKA and FKBdose-dependently potentiated the increase in ALT and AST induced by APAP(FIGS. 27A and 27B). Of note, one mouse with the treatment of thehighest dose of FKA and FKB in combination with APAP died ˜0.5-2 hoursbefore necropsy (i.e., 22 to 23.5 hours after the combined dose of APAPwith FKA and FKB). This was the only mouse among all the studies thatdied before necropsy. Its serum ALT and AST levels were the highestamong all mice (FIG. 27C), and 2-3 times higher than the next highestvalues. Histopathological examination revealed multifocal and coalescingacute centrilobular necrosis in the liver of this mouse (FIG. 27D, panelB), whereas livers from a control mouse (FIG. 27D, panel A) and a mousetreated with FKA and FKB alone (not shown) were histologically withinnormal limits. These data suggest that severe hepatotoxicity likelycontributed to its early death.

Discussion

Kava has demonstrated anxiolytic activity in the clinic and potentiallyreduces cancer risk in humans. On the other hand, kava usage has beenspeculated to be associated with rare but severe hepatotoxicity. Variousmechanisms have been proposed and different chemicals have beenpostulated with no confirmation. Given kava's global resurgence and thediverse chemical composition among current kava products, it is urgentand important to recapitulate kava's hepatotoxicity in an in vivo model,which can help identify the responsible chemicals and guide thedevelopment of strategies to minimize and ideally eradicate such anadverse potential.

The results from this study demonstrated that kava when administeredalone via gavage in C57BL/6 mice induced no adverse effect even at afairly high dose (500 mg/kg bodyweight daily) in a chronic manner, asreflected in mouse growth and serum levels of ALT and AST (FIG. 23).These results are consistent with the results from many early studies(DiSilvestro, et al. (2007) Food Chem. Toxicol. 45, 1293-1300; Guo, L.,(2009) Food Chem. Toxicol. 47, 433-442; Guo, et al. (2010) Food Chem.Toxicol. 48, 686-696; Sorrentino, et al. (2006) Phytomedicine 13,542-549). On the other hand, kava significantly potentiated thehepatotoxicity of APAP in C57BL/6 mice as indicated by the increase inserum ALT and AST, and the increased severity of liver lesions (FIG.24). The treatment regimen was designed to mimic potential circumstancesamong human kava users that kava would be consumed on a daily basiswhile other medications, APAP in this case, were used occasionally whenneeded. Since the majority of kava-associated hepatotoxic cases consumedother medications or dietary supplements concomitantly, the results fromthis study may have direct indication to the observed hepatotoxicityamong kava users. It remains to be determined whether kava usage canpotentiate the hepatotoxic risk of other medications or hepatotoxins,such as alcohol consumption. It also remains to be determined whetherother kava treatment regimens, such as prolonged kava usage or in afasted stage (recommended for traditional kava usage), may potentiateits hepatotoxic risk even at lower kava dosages.

With the C57BL/6 mouse model that captures kava's hepatotoxic risk invivo, the potential responsible compound(s) were investigated. Theresults demonstrated that a chalcone-based compound in kava, FKB,moderately potentiated APAP's hepatotoxicity while DHM, a representativeof kavalactones in kava, lacked such a risk when they were evaluated ata dose equivalent to kava at a dose of 500 mg/kg bodyweight (FIG. 26).As the kava product contains FKA, an analog of FKB, at similarabundance, the combination of FKA and FKB were evaluated, whichdose-dependently enhanced APAP-induced hepatotoxicity (FIG. 27). Indeedthe one mouse that died early, and which had the highest ALT and ASTlevels (FIG. 27C) reflecting extensive acute hepatocellular necrosis(FIG. 27D, panel B), was in the APAP co-treatment group at the highestdose of FKA and FKB. These data overall indicate that FKA and FKB arethe responsible compounds in kava that potentiate APAP-inducedhepatotoxicity while DHM is free of this risk. Besides FKA and FKB,flavokawain C (FKC) has been reported in other kava products (Lebot, etal. (2014) Food Chem. 151, 554-560) but was not detectable in the kavaproduct used in this study. FKC might be another compound responsiblefor hepatotoxicity.

The recent analysis of a set of kava products on the current marketdemonstrates that the abundance of FKA and FKB can vary ˜20 fold(Martin, et al. (2014) Plos One, 9(11): e111572.doi:10.1371/journal.pone.0111572). Similarly a recent study analyzed theabundance of FKA, FKB, and FKC in different kava cultivars (Lebot, etal. (2014) Food Chem. 151, 554-560). Cultivars not recommended fortraditional use were found to contain higher abundance of FKA, FKB, andFKC than the traditionally consumed cultivars (Lebot, et al. (2014) FoodChem. 151, 554-560). Further studies therefore are warranted to evaluatewhether cultivars or kava products with higher content of FKA, FKB, andFKC would impose a higher hepatotoxic risk. Future studies are alsoneeded to elucidate the molecular mechanisms of the observedhepatotoxicity enhancement, such as the depletion of glutathione (Zhou,et al. (2010) FASEB J. 24, 4722-4732). Such knowledge will help guidethe preparation of kava products for human use with higher healthbenefit and minimal adverse effects.

Example 6 Dihydromethysticin Effectively Blocks2-Amino-1-Methyl-6-Phenylimidazo(4,5-b)Pyridine (PhIP) andBenzo(a)Pyrene (BaP)-Induced DNA Damage Introduction

The goal of this study was to explore the potential scope ofdihydromethysticin in blocking/reducing different carcinogen-induced DNAdamage and detoxifying such carcinogens.2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of themost abundant heterocyclic amines in cooked meats (Ni W, (2008) Journalof agricultural and food chemistry. 56(1):68-78.) and possibly the mostpotent carcinogen among them, particularly for colon cancer, prostatecancer and breast cancer (Nishikawa A, (2005). Toxicological sciences:an official journal of the Society of Toxicology. 84(2):243-8.).Benzo(a)pyrene (BaP) is a representative polyaromatic hydrocarbon-basedcarcinogen in cigarette smoke and is present in various industrial/motorexhaust/waste. BaP is a human carcinogen to lung, stomach and othertissues. A rodent liver cancer cell line was used in these studies.Briefly, the hepe1c1c7 cells were co-treated with dihydromethysticin (1,5, and 25 μM) and PhIP (10 μM) for 24 hours with DNA isolated andPhIP-based DNA damage quantified (FIG. 28A). It is clear thatdihydromethysticin effectively and dose-dependently reduced PhIP-inducedDNA damage, demonstrating its efficacy in blocking carcinogen-inducedDNA damage and indicating its detoxifying function and cancerchemopreventive potential against colon, prostate and breast cancer.Similarly the cells were co-treated with dihydromethysticin (1, 5, and25 μM) and BaP (0.3 μM) for 3 hours with DNA isolated and BaP-based DNAdamage quantified (FIG. 28B). Dihydromethysticin effectively anddose-dependently reduced BaP-induced DNA damage, demonstrating itsefficacy in blocking carcinogen-induced DNA damage and indicating itsdetoxifying function and cancer chemopreventive potential against lungcancer and stomach cancer.

Example 7

The following illustrate representative pharmaceutical dosage forms,containing ‘Compound X’, for therapeutic or prophylactic use in humans.As described herein, Compound X may represent a compound describedherein, for example, such as dihydromethysticin, methysticin,dihydrokavain, kavain, desmethoxyyangonin and 11-methoxyyangonin.

(i) Tablet 1 mg/tablet Compound X = 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0

(ii) Tablet 2 mg/tablet Compound X = 20.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0500.0

(iii) Capsule mg/capsule Compound X = 10.0 Colloidal silicon dioxide 1.5Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0

(iv) Injection 1 (1 mg/ml) mg/ml Compound X = (free acid form) 1.0Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodiumchloride 4.5 1.0N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mg/ml) mg/ml Compound X = (free acid form) 10.0Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethyleneglycol 400 200.0 1.0N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(vi) Aerosol mg/can Compound X = 20.0 Oleic acid 10.0Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0Dichlorotetrafluoroethane 5,000.0

(vii) Aerosol 1 mg/can Compound X = 10.0 Oleic acid 10.0Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0Dichlorotetrafluoroethane 5,000.0

(viii) Aerosol 2 mg/can Compound X = 5.0 Oleic acid 10.0Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0Dichlorotetrafluoroethane 5,000.0

(ix) Aerosol 3 mg/can Compound X = 2.0 Oleic acid 10.0Trichloromonofluoromethane 5,000.0 Dichlorodifluoromethane 10,000.0Dichlorotetrafluoroethane 5,000.0

The above formulations may be obtained by conventional procedures wellknown in the pharmaceutical art.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A method, comprising: a) combining an ethanolickava extract and silica gel to provide a mixture; b) evaporating themixture to provide a silica gel having kava residue adsorbed thereon; c)loading the silica gel having kava residue adsorbed thereon on achromatography column to provide a kava-adsorbed silica gel column; d)eluting the kava-adsorbed silica gel column with a solvent system toprovide a first kava extract fraction, a second kava extract fraction,and a third kava extract fraction, wherein the first kava extractfraction consists essentially of non-polar compounds, includingflavokawains, the second kava extract fraction consists essentially ofkavalactones and flavanones, and the third kava extract fractionconsists essentially of polar compounds.
 2. The method of claim 1,wherein step d) comprises eluting with 28% ethyl acetate (EA) and 72%hexane (Hex) 5 column volumes (CV), followed by 90% EA and 10% Hex 4.1CV, and then 35% MeOH and 65% EA 5.5 CV.
 3. The method of claim 1,wherein the weight ratio of kava residue to the total weight of silicagel having kava residue adsorbed thereon is 0.3±0.2.
 4. A second kavaextract fraction prepared by the method of any one of claims 1-3.
 5. Acomposition comprising 11-methoxyyangonin and/or flavanone:

and at least one compound selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain anddesmethoxyyangonin, wherein the composition is substantially free offlavokawain B and/or flavokawain A.
 6. The composition of claim 5,wherein the at least one compound is dihydromethysticin.
 7. Thecomposition of claim 5, wherein at least two compounds are selected fromthe group consisting of dihydromethysticin, methysticin, dihydrokavain,kavain and desmethoxyyangonin, wherein the composition is substantiallyfree of flavokawain B and/or flavokawain A.
 8. The composition of claim5, wherein at least three compounds are selected from the groupconsisting of dihydromethysticin, methysticin, dihydrokavain, kavain anddesmethoxyyangonin, wherein the composition is substantially free offlavokawain B and/or flavokawain A.
 9. The composition of claim 5,wherein at least four compounds are selected from the group consistingof dihydromethysticin, methysticin, dihydrokavain, kavain anddesmethoxyyangonin, wherein the composition is substantially free offlavokawain B and/or flavokawain A.
 10. The composition of any one ofclaims 5-9, wherein the composition comprises 11-methoxyyangonin. 11.The composition of any one of claims 5-9, wherein the compositioncomprises flavanone:


12. The composition of any one of claims 5-9, wherein the compositioncomprises 11-methoxyyangonin and flavanone:


13. The composition of any one of claims 5-12, substantially free offlavokawain A.
 14. The composition of any one of claims 5-13,substantially free of bornyl ester of 3,4-methylenedioxy cinnamic acid:


15. The composition of any one of claims 5-14, substantially free ofbornyl ester of cinnamic acid:


16. The composition of any one of claims 5-15, substantially free offlavanone:


17. The composition of any one of claims 5-16, substantially free ofpinostrobin.
 18. The composition of any one of claims 5-17,substantially free of flavokawain B.
 19. A composition comprising atleast one compound selected from the group consisting ofdihydromethysticin, wherein the weight percent of dihydromethysticin inthe composition is about 20 to 99%; methysticin, wherein the weightpercent of methysticin in the composition is about 10 to 99%;dihydrokavain, wherein the weight percent of dihydrokavain in thecomposition is about 40 to 99%; kavain, wherein the weight percent ofkavain in the composition is about 40 to 99%; desmethoxyyangonin,wherein the weight percent of desmethoxyyangonin in the composition isabout 30 to 99%; and 11-methoxyyangonin, wherein the weight percent of11-methoxyyangonin in the composition is about 20 to 99%.
 20. Thecomposition of claim 19, wherein the at least one compound isdihydromethysticin, and wherein the weight percent of dihydromethysticinin the composition is about 20 to 99%.
 21. The composition of claim 19,wherein the at least one compound is kavain, and wherein the weightpercent of kavain in the composition is 40 to 99%.
 22. The compositionof claim 19, wherein the at least one compound is methysticin, andwherein the weight percent of methysticin in the composition is 10 to99%.
 23. The composition of claim 19, wherein the at least one compoundis dihydrokavain, and wherein the weight percent of dihydrokavain in thecomposition is 40 to 99%.
 24. The composition of claim 19, wherein theat least one compound is desmethoxyyangonin, and wherein the weightpercent of desmethoxyyangonin in the composition is 30 to 99%.
 25. Thecomposition of claim 19, wherein the at least one compound is11-methoxyyangonin, and wherein the weight percent of 11-methoxyyangoninin the composition is 20 to 99%.
 26. The composition of claim 19,substantially free of methysticin.
 27. The composition of any one ofclaims 19-26, further comprising flavanone:

and wherein the weight percent of the flavanone in the composition isabout 20 to 99%.
 28. The composition of any one of claims 19-27,substantially free of flavokawain B.
 29. The composition of any one ofclaims 19-28, substantially free of flavokawain A.
 30. The compositionof any one of claims 19-29, substantially free of bornyl ester of3,4-methylenedioxy cinnamic acid:


31. The composition of any one of claims 19-30, substantially free ofbornyl ester of cinnamic acid:


32. The composition of any one of claims 19-31, substantially free offlavanone:


33. The composition of any one of claims 19-32, substantially free ofpinostrobin.
 34. The composition of any one of claims 19-33, wherein thecomposition is a kava extract.
 35. A kava extract comprising11-methoxyyangonin and/or flavanone:

and at least one compound selected from the group consisting ofdihydromethysticin, methysticin, dihydrokavain, kavain anddesmethoxyyangonin, wherein the extract is substantially free offlavokawain B and/or flavokawain A.
 36. The kava extract of claim 35,wherein the at least one compound is dihydrokavain.
 37. The kava extractof claim 35, wherein the at least one compound is kavain.
 38. The kavaextract of claim 35, wherein the at least one compound is methysticin.39. The kava extract of claim 35, wherein the at least one compound isdihydromethysticin.
 40. The kava extract of claim 35, wherein the atleast one compound is desmethoxyyangonin.
 41. The kava extract of anyone of claims 35-40, substantially free of flavokawain B.
 42. The kavaextract of any one of claims 35-41, substantially free of flavokawain A.43. The kava extract of any one of claims 35-42, substantially free ofbornyl ester of 3,4-methylenedioxy cinnamic acid:


44. The kava extract of any one of claims 33-40, substantially free ofbornyl ester of cinnamic acid:


45. The kava extract of any one of claims 35-44, substantially free offlavanone:


46. The kava extract of any one of claims 35-45, substantially free ofpinostrobin.
 47. A kava extract consisting essentially ofdihydromethysticin, 11-methoxyyangonin, desmethoxyyangonin,dihydrokavain, kavain, methysticin and flavanone:


48. The composition or kava extract of any one of claims 4-47, whereinthe composition or extract is suitable for ingestion by a mammal (e.g.,a human).
 49. The composition or kava extract of any one of claims 4-48,wherein the composition or extract is formulated in a tablet, capsule,powder, spray, chewing gum, cream, gel, stent, inhalable, patch,nano-emulsion or liquid.
 50. A pharmaceutical composition comprising acomposition or kava extract as described in any one of claims 4-49 and apharmaceutically acceptable carrier.
 51. A method for treating orpreventing cancer in a mammal in need of such treatment comprising,administering to the mammal dihydromethysticin and a carrier, whereinthe dihydromethysticin is substantially free of other kava extractcomponents.
 52. A composition comprising dihydromethysticin and acarrier for the prophylactic or therapeutic treatment of cancer in amammal, wherein the dihydromethysticin is substantially free of otherkava extract components.
 53. The method of claim 51 or the compositionof claim 52, wherein the other kava extract components are selected fromthe group consisting of 11-methoxyyangonin, desmethoxyyangonin,dihydrokavain, kavain, methysticin, pinostrobin, flavokawain B,flavokawain A,


54. A method for treating or preventing cancer in a mammal in need ofsuch treatment comprising, administering to the mammal methysticin and acarrier, wherein the methysticin is substantially free of other kavaextract components.
 55. A composition comprising methysticin and acarrier for the prophylactic or therapeutic treatment of cancer in amammal, wherein the methysticin is substantially free of other kavaextract components.
 56. The method of claim 54 or the composition ofclaim 55, wherein the other kava extract components are selected fromthe group consisting of 11-methoxyyangonin, desmethoxyyangonin,dihydrokavain, kavain, dihydromethysticin, pinostrobin, flavokawain B,flavokawain A,


57. A method for treating or preventing cancer in a mammal in need ofsuch treatment comprising, administering to the mammal dihydrokavain anda carrier, wherein the dihydrokavain is substantially free of other kavaextract components.
 58. A composition comprising dihydrokavain and acarrier for the prophylactic or therapeutic treatment of cancer in amammal, wherein the dihydrokavain is substantially free of other kavaextract components.
 59. The method of claim 57 or the composition ofclaim 58, wherein the other kava extract components are selected fromthe group consisting of 11-methoxyyangonin, desmethoxyyangonin,dihydromethysticin, kavain, methysticin, pinostrobin, flavokawain B,flavokawain A,


60. A method for treating or preventing cancer in a mammal in need ofsuch treatment comprising, administering to the mammal kavain and acarrier, wherein the kavain is substantially free of other kava extractcomponents.
 61. A composition comprising kavain and a carrier for theprophylactic or therapeutic treatment of cancer in a mammal, wherein thekavain is substantially free of other kava extract components.
 62. Themethod of claim 60 or the composition of claim 61, wherein the otherkava extract components are selected from the group consisting of11-methoxyyangonin, desmethoxyyangonin, dihydrokavain,dihydromethysticin, methysticin, pinostrobin, flavokawain B, flavokawainA,


63. A method for treating or preventing cancer in a mammal in need ofsuch treatment comprising, administering to the mammaldesmethoxyyangonin and a carrier, wherein the desmethoxyyangonin issubstantially free of other kava extract components.
 64. A compositioncomprising desmethoxyyangonin and a carrier for the prophylactic ortherapeutic treatment of cancer in a mammal, wherein thedesmethoxyyangonin is substantially free of other kava extractcomponents.
 65. The method of claim 63 or the composition of claim 64,wherein the other kava extract components are selected from the groupconsisting of 11-methoxyyangonin, dihydromethysticin, dihydrokavain,kavain, methysticin, pinostrobin, flavokawain B, flavokawain A,


66. A method for treating or preventing cancer in a mammal in need ofsuch treatment comprising, administering to the mammal11-methoxyyangonin and a carrier, wherein the 11-methoxyyangonin issubstantially free of other kava extract components.
 67. A compositioncomprising 11-methoxyyangonin and a carrier for the prophylactic ortherapeutic treatment of cancer in a mammal, wherein the11-methoxyyangonin is substantially free of other kava extractcomponents.
 68. The method of claim 66 or the composition of claim 67,wherein the other kava extract components are selected from the groupconsisting of desmethoxyyangonin, dihydromethysticin, dihydrokavain,kavain, methysticin, pinostrobin, flavokawain B, flavokawain A,


69. The method of any one of claims 51, 54, 57, 60, 63 and 66 or thecomposition of any one of claims 52, 55, 58, 61, 64 and 67, wherein theother kava extract components are selected from the group consisting ofpinostrobin, flavokawain B, flavokawain A,


70. A method for treating or preventing cancer in a mammal in need ofsuch treatment comprising, administering to the mammal a composition orkava extract as described in any one of claims 4-50.
 71. A compositionor kava extract as described in any one of claims 4-50 for theprophylactic or therapeutic treatment of cancer in a mammal.
 72. The useof a composition or kava extract as described in any one of claims 4-50to prepare a medicament for treating or preventing cancer in a mammal.73. The method, composition or use of any one of claims 51-72, whereinthe cancer is lung cancer, prostate cancer, skin cancer, melanoma,genitourinary cancer, colon and rectum cancer, breast cancer, ovarycancer, esophagial cancer, pancreatic cancer, urinary bladder cancer,cervical cancer, liver cancer, kidney and renal cancer, head and neckcancer, brain cancer or various hematological cancers.
 74. A method forpreventing tumorigenesis, reducing DNA damage, reducing protein damageand/or detoxifying physical or chemical carcinogens in a mammal in needof such treatment comprising, administering to the mammaldihydromethysticin and a carrier, wherein the dihydromethysticin issubstantially free of other kava extract components.
 75. A compositioncomprising dihydromethysticin and a carrier for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal, wherein thedihydromethysticin is substantially free of other kava extractcomponents.
 76. The method of claim 74 or the composition of claim 75,wherein the other kava extract components are selected from the groupconsisting of 11-methoxyyangonin, desmethoxyyangonin, dihydrokavain,kavain, methysticin, pinostrobin, flavokawain B, flavokawain A,


77. A method for preventing tumorigenesis, reducing DNA damage, reducingprotein damage and/or detoxifying physical or chemical carcinogens in amammal in need of such treatment comprising, administering to the mammalmethysticin and a carrier, wherein the methysticin is substantially freeof other kava extract components.
 78. A composition comprisingmethysticin and a carrier for preventing tumorigenesis, reducing DNAdamage, reducing protein damage and/or detoxifying physical or chemicalcarcinogens in a mammal, wherein the methysticin is substantially freeof other kava extract components.
 79. The method of claim 77 or thecomposition of claim 78, wherein the other kava extract components areselected from the group consisting of 11-methoxyyangonin,desmethoxyyangonin, dihydrokavain, kavain, dihydromethysticin,pinostrobin, flavokawain B, flavokawain A,


80. A method for preventing tumorigenesis, reducing DNA damage, reducingprotein damage and/or detoxifying physical or chemical carcinogens in amammal in need of such treatment comprising, administering to the mammaldihydrokavain and a carrier, wherein the dihydrokavain is substantiallyfree of other kava extract components.
 81. A composition comprisingdihydrokavain and a carrier for preventing tumorigenesis, reducing DNAdamage, reducing protein damage and/or detoxifying physical or chemicalcarcinogens in a mammal, wherein the dihydrokavain is substantially freeof other kava extract components.
 82. The method of claim 80 or thecomposition of claim 81, wherein the other kava extract components areselected from the group consisting of 11-methoxyyangonin,desmethoxyyangonin, dihydromethysticin, kavain, methysticin,pinostrobin, flavokawain B, flavokawain A,


83. A method for preventing tumorigenesis, reducing DNA damage, reducingprotein damage and/or detoxifying physical or chemical carcinogens in amammal in need of such treatment comprising, administering to the mammalkavain and a carrier, wherein the kavain is substantially free of otherkava extract components.
 84. A composition comprising kavain and acarrier for preventing tumorigenesis, reducing DNA damage, reducingprotein damage and/or detoxifying physical or chemical carcinogens in amammal, wherein the kavain is substantially free of other kava extractcomponents.
 85. The method of claim 83 or the composition of claim 84,wherein the other kava extract components are selected from the groupconsisting of 11-methoxyyangonin, desmethoxyyangonin, dihydrokavain,dihydromethysticin, methysticin, pinostrobin, flavokawain B, flavokawainA,


86. A method for preventing tumorigenesis, reducing DNA damage, reducingprotein damage and/or detoxifying physical or chemical carcinogens in amammal in need of such treatment comprising, administering to the mammaldesmethoxyyangonin and a carrier, wherein the desmethoxyyangonin issubstantially free of other kava extract components.
 87. A compositioncomprising desmethoxyyangonin and a carrier for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal, wherein thedesmethoxyyangonin is substantially free of other kava extractcomponents.
 88. The method of claim 86 or the composition of claim 87,wherein the other kava extract components are selected from the groupconsisting of 11-methoxyyangonin, kavain, dihydrokavain,dihydromethysticin, methysticin, pinostrobin, flavokawain B, flavokawainA,


89. A method for preventing tumorigenesis, reducing DNA damage, reducingprotein damage and/or detoxifying physical or chemical carcinogens in amammal in need of such treatment comprising, administering to the mammal11-methoxyyangonin and a carrier, wherein the 11-methoxyyangonin issubstantially free of other kava extract components.
 90. A compositioncomprising 11-methoxyyangonin and a carrier for preventingtumorigenesis, reducing DNA damage, reducing protein damage and/ordetoxifying physical or chemical carcinogens in a mammal, wherein the11-methoxyyangonin is substantially free of other kava extractcomponents.
 91. The method of claim 89 or the composition of claim 90,wherein the other kava extract components are selected from the groupconsisting of desmethoxyyangonin, kavain, dihydrokavain,dihydromethysticin, methysticin, pinostrobin, flavokawain B, flavokawainA,


92. The method of any one of claims 74, 77, 80, 83, 86 and 89 or thecomposition of any one of claims 75, 78, 81, 84, 87 and 90, wherein theother kava extract components are selected from the group consisting ofpinostrobin, flavokawain B, flavokawain A,


93. A method for preventing tumorigenesis, reducing DNA damage, reducingprotein damage and/or detoxifying physical or chemical carcinogens in amammal in need of such treatment comprising, administering to the mammala composition or kava extract as described in any one of claims 4-50.94. A composition or kava extract as described in any one of claims 4-50for preventing tumorigenesis, reducing DNA damage, reducing proteindamage and/or detoxifying physical or chemical carcinogens in a mammal.95. The use of a composition or kava extract as described in any one ofclaims 4-50 to prepare a medicament for preventing tumorigenesis,reducing DNA damage, reducing protein damage and/or detoxifying physicalor chemical carcinogens in a mammal.
 96. The method, composition or useof any one of claims 74-95, wherein the DNA damage is a DNA adduct. 97.The method of claim 96, wherein the DNA adduct is selected from BaP,PhIP, POB and PHB adducts.
 98. The method of claim 96, wherein the DNAadduct is a O⁶-methylguanine DNA adduct.